U.S. patent application number 13/414841 was filed with the patent office on 2012-07-05 for uses of yerba santa.
Invention is credited to Maxim Golovkin, Hilary Koprowski, Natalia Pogrebnyak.
Application Number | 20120174264 13/414841 |
Document ID | / |
Family ID | 41505693 |
Filed Date | 2012-07-05 |
United States Patent
Application |
20120174264 |
Kind Code |
A1 |
Koprowski; Hilary ; et
al. |
July 5, 2012 |
Uses of Yerba Santa
Abstract
Methods of in vitro propagation of plants of the genus
Eriodictyon are described, including, in particular embodiments,
plants of the species E. californicum, E. trichocalyx and E.
sessilifolium. Methods of producing transgenic plants of the genus
Eriodictyon are also described, along with methods of producing
recombinant proteins in such plants. Compositions and methods for
administering recombinant proteins produced in these plants are
also described.
Inventors: |
Koprowski; Hilary;
(Wynnewood, PA) ; Pogrebnyak; Natalia; (Highland
Park, NJ) ; Golovkin; Maxim; (Feasterville,
PA) |
Family ID: |
41505693 |
Appl. No.: |
13/414841 |
Filed: |
March 8, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12487942 |
Jun 19, 2009 |
8148609 |
|
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13414841 |
|
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61074376 |
Jun 20, 2008 |
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Current U.S.
Class: |
800/298 ;
435/183; 435/469; 530/350; 530/351; 530/380; 530/387.1; 530/396;
530/399 |
Current CPC
Class: |
C12N 15/8257 20130101;
A61K 39/12 20130101; A61K 2039/517 20130101; A61K 39/145 20130101;
C12N 2760/16134 20130101; C12N 15/8205 20130101; A61K 2039/5258
20130101; A61K 2039/55544 20130101; A61K 2039/542 20130101; A61K
2039/543 20130101; C12N 15/8258 20130101; A01H 4/005 20130101 |
Class at
Publication: |
800/298 ;
435/469; 530/350; 530/387.1; 530/399; 435/183; 530/380; 530/351;
530/396 |
International
Class: |
A01H 5/00 20060101
A01H005/00; C07K 14/00 20060101 C07K014/00; C07K 16/00 20060101
C07K016/00; C07K 14/575 20060101 C07K014/575; C07K 14/405 20060101
C07K014/405; C07K 14/435 20060101 C07K014/435; C07K 14/555 20060101
C07K014/555; C07K 14/005 20060101 C07K014/005; C07K 14/11 20060101
C07K014/11; C12N 15/82 20060101 C12N015/82; C12N 9/00 20060101
C12N009/00 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with United States government
support awarded by the United States Department of Agriculture,
Grant Number SCA 58-1275-4-303. The United States has certain
rights in this invention.
Claims
1. A transgenic plant of the genus Eriodictyon.
2. The transgenic plant of claim 1, wherein the plant is selected
from the group consisting of E. californicum, E. trichocalyx and E.
sessilifolium.
3. The transgenic plant of claim 1, which expresses a recombinant
protein.
4. The transgenic plant of claim 3 wherein the recombinant protein
comprises an avian influenza HA1 antigen.
5. A method for transforming tissue from a plant of the genus
Eriodictyon comprising the steps of: inoculating leaf tissue from
an Eriodictyon plant with an Agrobacterium suspension diluted to
about OD.sub.600 0.005 to 0.5, the Agrobacterium containing at
least one genetic component encoding a desired protein capable of
being transferred to the transformable plant tissue and of
directing the expression of the desired protein in the plant
tissue; co-cultivating the plant tissue with the Agrobacterium;
transferring the plant tissue to recovery media containing an
antibiotic for eliminating the Agrobacterium; and selecting
transformed plant tissue.
6. The method of claim 5 wherein the plant species is selected from
the group consisting of Eriodictyon californicum, Eriodictyon
trichocalyx and Eriodictyon sessilifolium.
7. The method of claim 5 wherein the Agrobacterium suspension is
diluted to about OD.sub.600 0.03.
8. The method of claim 5 wherein the co-cultivating of the plant
tissue with the Agrobacterium suspension is for about 1 to 4 days
at about 20 to 25.degree. C.
9. The method of claim 8 wherein the co-cultivating of the plant
tissue with the Agrobacterium suspension is for about 2 days.
10. A method for producing a recombinant protein in a transgenic
plant of the genus Eriodictyon comprising the steps of: providing a
transgenic plant that has been regenerated from a transformed plant
cell or tissue of the genus Eriodictyon and that expresses a
recombinant protein; and recovering the recombinant protein
expressed in the transgenic plant.
11. The method of claim 10 wherein the recovery step further
comprises obtaining an extract of the transgenic plant.
12. The method of claim 10 wherein the recovery step further
comprises harvesting material from at least a portion of the
transgenic plant.
13. The method of claim 12 wherein the at least a portion of the
harvested material is edible.
14. The method of claim 10 wherein the plant cell or tissue is
transformed using an Agrobacterium system.
15. The method of claim 14 wherein the plant cell or tissue is
transformed using the method of claim 5.
16. The method of claim 10 wherein the recombinant protein is
selected from the group consisting of antigens, microbicides,
antibodies, hormones, enzymes, blood components, interferons, and
anticoagulants.
17. The method of claim 16 wherein the antigen comprises a viral
protein.
18. The method of claim 17 wherein the protein comprises an avian
influenza HA1 antigen.
19. The method of claim 16 wherein the microbicide comprises an
antiretroviral.
20. The method of claim 19 wherein the antiretroviral comprises
griffithsin.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority from U.S. Non-Provisional
application Ser. No. 12/487,942 filed on Jun. 19, 2009, which
claims priority from U.S. Provisional Application No. 61/074,376
filed on Jun. 20, 2008, the disclosures of which are herein
incorporated by reference.
BACKGROUND OF THE INVENTION
[0003] The medicinal plant Yerba Santa, member of the Waterleaf
Family, genus Eriodictyon, has a long tradition of use. Yerba Santa
(including Eriodictyon californicum, Eriodictyon trichocalyx and
other related species) has been used in treating respiratory
conditions, including colds, cough, asthma and bronchitis. This
herb has also been found effective for a number of other symptoms
including gastrointestinal disorders, fatigue, rheumatism, and
allergies. Biochemical analyses have confirmed Yerba Santa to have
flavonoids that show promise as anti-carcinogens (Liu Y L, Ho D K,
Cassady J M, Cook V M, Baird W M (1992) J Nat Prod 55:357-363).
[0004] For medical purposes, Yerba Santa has been used as either a
dry herb or an extract. (Heizer R F, Elsasser A B (1980) The
natural world of the California Indians, University of California
Press, Berkeley & Los Angeles, Calif. 271 pp.). Many herbal
stores carry different products containing Yerba Santa such as, for
example, leaf powder, extracts, leaf tea, and cream. The fluid
Yerba Santa extracts have been used in food, beverages,
pharmaceuticals and cosmetics.
[0005] Yerba Santa is a perennial evergreen shrub (1-2 meters) that
grows in dry, hilly areas of California and Northern Mexico. During
dry months, the leaves become hard and resinous in order to hold
and conserve water. When applied to mucosal surfaces, the herb
preparation holds the aqueous component in contact with cells,
reestablishing mucopolysaccharides. It is hypothesized that this
property may facilitate the adherence to the mucosa of compositions
such as, for example, pharmaceutical agents.
[0006] In recent years, there has been an increased interest in
mass multiplication of this unique plant. In vitro culture
techniques have been used successfully for large scale production
of many medicinally important plant species. However, methods for
in vitro propagation and tissue culture techniques for Yerba Santa
have not previously been described.
[0007] Plant biotechnology has provided successful tissue culture
and transformation technologies for a variety of plants. However,
the use of biotechnological tools in medicinal plant science has
been very limited as compared to other crops. Nevertheless, in
recent years such techniques have been developed for number of
important medicinal plants such as Ginkgo biloba, Digitalis lanata,
Artenisia annua, Papaver somniferum, Camptotheca acuminate,
Ophiorrhiza prostrate, and Mentha piperita. Although Yerba Santa is
a very important medicinal plant, there have been no techniques
described for in vitro propagation, cell culture cultivation,
regeneration and transformation of this perennial shrub.
[0008] Modern plant biotechnology has opened new avenues for
producing recombinant molecules, including, but not limited to,
vaccines (Hansson M, Nygren P A & Stahl S (2000) Biotechnol
Appl Biochem 32:95-107; Daniell H, Streatfield S J & Wycoff K
(2001) Trends Plant Sci 6:219-226; Ma J K C, Drake P M W &
Christou P (2003) Nat Rev Genet. 4:794-805; Koprowski H (2005)
Vaccine 23:1757-1763; Pogrebnyak N., Golovkin M., Andrianov V.,
Spitsin S., Smirnov Y., Egolf R., Koprowski H (2005) Proc Natl Acad
Sci USA 102: 9062-9067; and Golovkin M., Spitsin S., Andrianov V.,
Smirnov Y., Xiao Y., Pogrebnyak N., Markley K., Brodzik R., Gleba
Y., Isaacs S N, Koprowski H. Proc Natl Acad Sci USA (2007), 104:
6864-6869; Goldstein D A, Thomas J A (2004) QJ Med 97:705-716) or
microbicides (O'Keefe B. et al. Proc Natl Acad Sci USA (2009), 106:
6099-6104). This approach has become an attractive alternative to
other technologies since it is associated with low production cost,
overall safety, and scalability potential. A potential benefit of
using plants for vaccine production is the possibility of applying
preparations directly to bodily surfaces such as, for example,
mucosal surfaces (Goldstein D A, Thomas J A (2004) QJ Med
97:705-716; Giddings G, Allison G, Brooks D, Carter A (2005) Nat
Biotechnol 18:1151-1155; Pogrebnyak N. Markley K., Smirnov Y.,
Brodzik R., Bandurska K., Koprowski H., Golovkin M (2006) Plant
Sci. 171: 677-685); Golovkin M., Spitsin S., Andrianov V., Smirnov
Y., Xiao Y., Pogrebnyak N., Markley K., Brodzik R., Gleba Y.,
Isaacs S N, Koprowski H. Proc Natl Acad Sci USA (2007), 104:
6864-6869; O'Keefe B. et al. Proc Natl Acad Sci USA (2009), 106:
6099-6104).
BRIEF SUMMARY OF INVENTION
[0009] The invention relates to a transgenic plant of the genus
Eriodictyon, in particular plants of the species E. californicum,
E. trichocalyx or E. sessilifolium. In one aspect, the transgenic
plant expresses a recombinant protein selected from the group
consisting of antigens, microbicides, antibodies, hormones,
enzymes, blood components, interferons, and anticoagulants. In a
particular embodiment, the antigen is a viral protein such as an
avian influenza HA1 antigen. In another embodiment, the microbicide
is an antiretroviral such as griffithsin.
[0010] In another embodiment, the present invention relates to a
method for transforming a plant tissue, particularly plant tissue
from a plant species of the genus Eriodictyon including the steps
of: inoculating a transformable plant tissue with an Agrobacterium
suspension, the Agrobacterium containing at least one genetic
component encoding a desired protein capable of being transferred
to the transformable plant tissue and of directing the expression
of the desired protein in the plant tissue; co-cultivating the
plant tissue with the Agrobacterium; transferring the plant tissue
to recovery media containing an antibiotic for eliminating the
Agrobacterium; and selecting transformed plant tissue.
[0011] The invention also relates to a method for producing a
recombinant protein in a transgenic plant of the genus Eriodictyon
including the steps of: providing a transgenic plant that has been
regenerated from a transformed plant cell or tissue of the genus
Eriodictyon and that expresses a recombinant protein; and
recovering the protein expressed in the transgenic plant.
[0012] One embodiment of the present invention relates to a method
of delivering a recombinant protein to a subject including
providing harvested material from a transgenic plant of the genus
Eriodictyon that expresses a recombinant protein; and administering
the harvested material to the subject in an amount necessary to
deliver an effective amount of the recombinant protein. In a
particular aspect, the recombinant protein is an antigen and the
harvested material is administered in an amount sufficient to
induce an immune response in the subject. In another aspect, the
recombinant protein is a microbicide and the harvested material is
administered in an amount sufficient to provide a prophylactic
effect.
[0013] In another embodiment, the invention relates to a method of
propagating in vitro a plant of the genus Eriodictyon, the method
including the steps of: excising a stem segment of the plant; and
incubating the segment in a growth medium comprising a cytokinin;
whereby the segment produces a shoot. In another aspect, the method
further includes excising the shoot; and incubating the excised
shoot in medium comprising an auxin, whereby the shoot produces a
root.
[0014] In another embodiment, the method of propagating in vitro a
plant of the genus Eriodictyon further includes incubating the
shoot for at least three weeks, whereby the shoot produces a leaf;
cutting a segment from the leaf; placing the segment in a culture
medium comprising one or more of benzylaminopurine,
naphthaleneacetic acid or 2,4-dichlorophenoxyacetic acid; and
incubating the segment in the dark, whereby the segment develops
callus tissue.
[0015] In another aspect, the invention relates to a method of
producing a cell suspension culture of a plant of the genus
Eriodictyon including the steps of: excising a portion of the
callus tissue produced according to certain aspects of the
invention;
[0016] placing the callus tissue in a liquid medium comprising
2,4-dichlorophenoxyacetic acid to form a cell suspension; and
incubating the cell suspension in the dark while agitating the
medium.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIG. 1 illustrates in vitro propagation of Yerba Santa. (A)
Yerba Santa E. trichocalyx stem segments, 3 days on MS medium with
1 mg/l zeatin (B) E. trichocalyx stem segments produced multiple
shoots after 5 weeks on MS medium with 1 mg/l zeatin. (C) Root
formation in E. sessilifolium after 10 days on MS medium. (D) Root
induction in E. trichocalyx on MS medium with 1 mg/l IBA, view of
bottom of plastic culture box. (E) Root induction in E.
californicum on MS medium with 1 mg/l IBA. (F) Growth and
development of E. trichocalyx plant in vitro. (G) E. californicum
plant in a pot, 2 weeks after transfer. (H) E. trichocalyx plant in
a pot, 4 weeks after transfer.
[0018] FIG. 2 illustrates Yerba Santa callus and cell suspension
cultures. (A) Callus propagation on MS medium with 1 mg/l 2.4-D (E.
trichocalyx). (B) Callus propagation on MS medium with 1 mg/l 2,4-D
(E. californicum). (C) Cell suspension (E. trichocalyx), 1 day in
liquid MS medium with 0.5 mg/l 2,4-D. (D) Cell suspension (E.
trichocalyx) after 2 weeks in liquid MS medium with 0.5 mg/l
2,4-D.
[0019] FIG. 3 is a graph illustrating the growth rate of a Yerba
Santa E. trichocalyx cell suspension.
[0020] FIG. 4 is a graph illustrating shoot regeneration efficiency
in three Yerba Santa species. Regeneration efficiency is expressed
as the percent of explants producing shoots on MSR medium during 6
weeks.
[0021] FIG. 5 illustrates an assessment of regeneration and
transformation procedures for Yerba Santa (E. trichocalyx) (A)
Regeneration through callus formation; (B) Direct regeneration from
leaf explant on MSR medium; (C) Transgenic plant expressing Avian
flu antigen on MST-5 medium containing 100 mg/l kanamycin; (D)
Multiplication of transgenic line on MSP medium.
[0022] FIG. 6 illustrates a transgenic Yerba Santa (E. trichocalyx)
expressing Avian flu antigen. (A) Transgenic plant on root
induction medium; (B) Transgenic plant in a pot.
[0023] FIG. 7 is a Western blot analysis of transgenic Yerba Santa
plants showing expression of recombinant HA1 antigen. (A) SDS/PAGE;
(B) Western blot using HA1 antigen-specific antibodies; (C) Western
blos using c-Myc antibodies.
DETAILED DESCRIPTION OF THE INVENTION
[0024] Plants have emerged as modern efficient systems for
production and delivery of recombinant products, including, but not
limited to, microbicides and antigens. When applied on a mucosal
surface, preparations of this herb have a capacity to hold aqueous
components in contact with mucosal cells that may facilitate
adherence of pharmaceuticals to the mucosa. Accordingly, Yerba
Santa may be useful as a vehicle for effective delivery of
recombinant molecules, such as recombinant microbicides or
vaccines, to body surfaces including mucosal surfaces, including,
for example, intranasal and oral surfaces. A benefit of using
plants for production of recombinant drugs is that they allow
direct mucosal (and thus needle-free) administration of the
pharmaceutical. For mucosal delivery of a vaccine antigen to a
subject, the extended mucosal exposure to such a plant-based
vaccine may significantly increase immune response in the subject.
Microbicides reduce the infectivity of microbes, such as viruses or
bacteria, by averting infection at the mucosal surfaces.
Accordingly, a plant based delivery system that is directly applied
to mucosal surfaces is ideally suited for the administration of
microbicides.
[0025] Yerba Santa is suitable for human consumption, has been used
in medicine for centuries, and is widely used in the food industry.
According to certain aspects of the invention, modern plant
biotechnology techniques have been developed for this valuable
medicinal plant, including the development of transgenic Yerba
Santa plants. In certain embodiments, in vitro propagation,
regeneration and transformation systems are provided for Yerba
Santa species. In certain aspects, cell culture technologies are
provided for Yerba Santa that may offer economically favorable
methods for production of large amounts of recombinant
pharmaceuticals. For example, cell suspensions may be used to
produce large amounts of biopharmaceuticals in bioreactors under
controlled conditions to achieve uniform, high quality
products.
[0026] One aspect of the present invention is a transgenic plant;
preferably the plant is a member of the genus Eriodictyon.
Preferably, the plant is E. californicum, E. trichocalyx or E.
sessilifolium.
[0027] In certain embodiments, the invention relates to a
recombinant protein expressed by a transgenic plant according to
aspects of the invention.
[0028] As used herein, a "recombinant protein" means that the
protein, whether comprising a native or mutant primary amino acid
sequence, is obtained by expression of a gene carried by a
recombinant DNA molecule in a cell other than the cell in which
that gene and/or protein is naturally found. In other words, the
gene is heterologous to the host in which it is expressed. This
protein may include an entire (full-length) protein or a
polypeptide molecule, or may comprise a protein or polypeptide
fragment suitable for a particular purpose. Preferably, the
recombinant proteins expressed by the transgenic plant are suitable
for use as pharmaceuticals, including, but not limited to,
antigens, microbicides, antibodies, hormones, enzymes, blood
components, including, but not limited to, coagulation factors,
interferons, and anticoagulants.
[0029] As used herein, "antigen" may include a single antigen or a
plurality of antigens as long as at least one antigen is included
which, when administered in a sufficient amount, can induce an
immune response in a subject. The term "antigen" also includes any
portion of an antigen, e.g., the epitope, which can induce an
immune response. Preferably, the one or more antigens will produce
a sufficient immune response to confer resistance to infection upon
the recipient of the antigen. Examples of immunogenic or antigenic
molecules that may be useful include, without limitation, viral
antigens such as the entirety or portions of: Hepatitis virus B
surface antigen, Malaria parasite antigen, Influenza A H1N1
antigen, Rabies virus glycoprotein, Escherichia coli heat-labile
enterotoxin, Human rhinovirus 14 (HRV-14), human immunodeficiency
virus type (HIV-1) epitopes, Norwalk virus capsid protein,
Diabetes-associated autoantigen, Mink Enteritis Virus epitope, Foot
and mouth disease virus VP1 structural protein, Cholera toxin B
subunit, Human insulin-Cholera toxin B subunit fusion protein,
Human cytomegalovirus glycoprotein B, S. mutans, respiratory
syncytial virus antigens (F1, F2, G), tetanus toxin fragment C,
diphtheria toxin, S1 subunit of pertussis toxin and SARS
S-glycoprotein.
[0030] In certain embodiments, the protein expressed by the
transgenic plant comprises an antigen protein, preferably a viral
protein, more preferably an avian influenza HA1 antigen.
[0031] As used herein, "microbicide" refers to any compound or
substance whose purpose is to reduce the infectivity of microbes,
such as viruses or bacterial. Examples of microbicides that may be
useful include, without limitation, griffithsin, the fusion
inhibitor C52, RANTES analogue PSC-RANTES, and lectin cyanovirin-N
(CV-N).
[0032] In certain embodiments, the recombinant protein expressed by
the transgenic plant comprises a microbicide, preferably an
antiretroviral microbicide, more preferably an HIV entry inhibitor,
even more preferably griffithsin. As used herein, "griffithsin",
which has been shown to be a highly potent HIV entry inhibitor,
refers to a 121-amino-acid protein isolated from the red algae
Griffithsia or active mutants or fragments thereof. Griffithsin.
Mori T, O'Keefe B R, Sowder R C, et al. (2005), "Isolation and
characterization of griffithsin, a novel HIV-inactivating protein,
from the red alga Griffithsia sp", J. Biol. Chem. 280 (10):
9345-53.
[0033] Examples of antibodies that may be useful include, without
limitation, monoclonal antibodies (mAbs) and secretory IgA (sigA).
Antibody formats may include, without limitation, full-size, Fab
fragments, single-chain antibody fragments, bi-specific scFv
fragments, membrane anchored scFv, chimeric antibodies and
humanized antibodies.
[0034] Examples of hormones that may be useful include, but are not
limited to insulin, somatotropin, and erythropoietin.
[0035] Examples of enzymes that may be useful include, without
limitation, .alpha.-1-antitrypsin, aprotinin,
Antiotensin-1-converting enzyme, and Glucocerebrosidase
[0036] Examples of blood components that may be useful include,
without limitation, serum albumin, including human serum albumin,
and hemoglobin.
[0037] Examples of interferons that may be useful include, without
limitation, interferon-.alpha..
[0038] Examples of anticoagulants that may be useful include,
without limitation, protein C (serum protease) and hirudin.
[0039] In certain embodiments, the invention relates to methods for
producing recombinant proteins in transgenic plants comprising the
steps of: constructing a plasmid vector or a DNA fragment by
operably linking a DNA molecule comprising a sequence encoding a
protein to a promoter capable of directing the expression of the
protein in the plant; transforming a plant cell with the plasmid
vector or DNA fragment to create a transgenic plant cell; and
recovering the protein expressed in the plant cell for use.
Preferably, the plant is a member of the genus Eriodictyon.
Preferably, the plant is E. californicum, E. trichocalyx, or E.
sessilifolium. Preferably, the recombinant proteins are suitable
for use as pharmaceuticals, including, but not limited to,
antigens; microbicides; antibodies; hormones; enzymes; blood
components, including, but not limited to, coagulation factors;
interferons, and anticoagulants.
[0040] Exogenous DNA constructs used for transforming plant cells
will comprise the coding sequence of recombinant protein desired to
be expressed and usually other elements such as, but not limited to
introns, 5' and 3' untranslated regions, and promoters. In certain
embodiments, the utilization of a strong promoter, such as the
Rubisco promoter operably linked to the coding sequence of the
desired recombinant protein, provides large amounts of recombinant
protein in Yerba Santa leaves.
[0041] As is well known in the art, DNA constructs for use in
transforming plants and expressing a coding sequence typically also
comprise other regulatory elements in addition to a promoter, such
as but not limited to 3' untranslated regions (such as
polyadenylation sites), transit or signal peptides and marker
coding sequences elements.
[0042] During transformation, exogenous DNA may be introduced
randomly, i.e. at a non-specific location, in the plant genome. In
some cases, it may be useful to target an exogenous DNA insertion
in order to achieve site-specific integration, e.g. to replace an
existing gene sequence or region in the genome. In some other cases
it may be useful to target an exogenous DNA integration into the
genome at a predetermined site from which it is known that gene
expression occurs.
[0043] In practice DNA is introduced into only a small percentage
of target cells in any one experiment. Marker genes are used to
provide an efficient system for identification of those cells that
are stably transformed by receiving and integrating an exogenous
DNA construct into their genomes. Preferred marker genes provide
selective markers which confer resistance to a selective agent,
such as an antibiotic or herbicide. Potentially transformed cells
are exposed to the selective agent. In the population of surviving
cells will be those cells where, generally, the
resistance-conferring coding sequence has been integrated and
expressed at sufficient levels to permit cell survival. Cells may
be tested further to confirm stable integration of the exogenous
DNA. Useful selective marker genes include those conferring
resistance to antibiotics such as kanamycin (nptII), hygromycin B
(aph IV) and gentamycin (aac3 and aacC4) or resistance to
herbicides such as glufosinate (bar or pat) and glyphosate (EPSPS;
CP4).
[0044] Means for transforming plant cells are well known in the
art. Suitable methods include any method by which DNA can be
introduced into a cell, such as by Agrobacterium mediated
transformation, via bombardment by DNA coated particles, direct
delivery of DNA such as, for example, by PEG-mediated
transformation of protoplasts, by desiccation/inhibition-mediated
DNA uptake, by electroporation, or by agitation with silicon
carbide fibers.
[0045] In certain embodiments, the methods further comprise
regenerating a transgenic plant from the transgenic plant cell.
This step is performed prior to recovering a recombinant protein
expressed by the transgenic plant. In certain aspects, the methods
may comprise a recovery step that further comprises obtaining an
extract of the plant cell. In certain aspects, the methods may
comprise harvesting material from at least a portion of the
transgenic plant. In certain embodiments, at least a portion of the
material harvested from the transgenic plant is edible.
[0046] Preferably, the methods directed to transforming a plant
cell may comprise the use of an Agrobacterium system.
[0047] In certain embodiments of the invention, a recombinant
protein is expressed in a transgenic plant, at least a portion of
which plant is edible. In certain aspects, the recombinant protein
may be administered to a subject by oral administration, wherein a
portion of the plant expressing the recombinant protein is ingested
by the subject, such that an effective amount of the recombinant
protein is delivered to the subject.
[0048] The invention relates, in certain aspects to a method for
constructing a transgenic plant cell comprising the steps of:
constructing a plasmid vector or a DNA fragment by operably linking
a DNA molecule, the DNA molecule comprising a sequence encoding a
protein, to a promoter capable of directing the synthesis of the
protein in the plant; and transforming a plant cell with the
plasmid vector or DNA fragment; preferably, the plant is a member
of the genus Eriodictyon. In certain embodiments, the plant is
preferably E. californicum, E. trichocalyx, or E.
sessilifolium.
[0049] Preferably, the method of transforming the plant cell
comprises the use of an Agrobacterium system.
[0050] In certain embodiments, the method may further comprise
regenerating transgenic plants from the transgenic plant cell.
[0051] In certain aspects, the present invention relates to several
factors that influence efficiency of Agrobacterium-mediated
transformation. For example, the invention relates to a method for
transforming a plant tissue comprising the steps of: inoculating a
transformable plant tissue with an Agrobacterium suspension diluted
to about OD.sub.600 0.005 to 0.5, the Agrobacterium containing at
least one genetic component encoding a desired protein capable of
being transferred to the transformable plant tissue and of
directing the expression of the desired protein in the plant
tissue; co-cultivating the plant tissue with the Agrobacterium;
transferring the plant tissue to recovery media containing an
antibiotic for eliminating the Agrobacterium; and selecting
transformed plant tissue. In certain embodiments, the plant tissue
is from a plant species of the genus Eriodictyon, preferably
Eriodictyon californicum, Eriodictyon trichocalyx and Eriodictyon
sessilifolium.
[0052] As used herein, "OD.sub.600" means the optical density
measure at a wavelength of 600 nanometers. In a preferred
embodiment, the inoculating step is performed with an Agrobacterium
suspension diluted to about OD.sub.600 0.01 to 0.05, more
preferably about OD.sub.600 0.03.
[0053] In certain embodiments, the length or temperature of the
steps in the transforming method must be precisely monitored to
achieve transformed plants. For example, the inoculating step may
be performed for about 1 to 30 minutes, preferably for about 5 to
15 minutes, and more preferably for about 10 minutes. Likewise, the
co-cultivating step may be performed for about 1 to 4 days,
preferably for about 2 days, at about 20 to 25.degree. C. Finally,
the plant tissue may remain in the recovery media for about 5 to 20
days, more preferably for about 10 days.
[0054] In other embodiments, the method for transforming a plant
tissue further comprises transferring the plant tissue to media
containing successively higher concentrations of a selective agent.
Typical selective agents include but are not limited to antibiotics
such as kanamycin, geneticin, paromomycin or other chemicals such
as glyphosate. In one embodiment, the selective agent is kanamycin
and the genetic component of the Agrobacterim is capable of
conferring kanamycin resistance to the plant tissue.
[0055] The present invention, in certain aspects, also relates to
methods for producing a recombinant protein in a transgenic plant
of the genus Eriodictyon comprising the steps of: providing a
transgenic plant that has been regenerated from a transformed plant
cell or tissue of the genus Eriodictyon and that expresses a
recombinant protein; and recovering the protein expressed in the
transgenic plant. Preferably, the plant is from the species of E.
californicum, E. trichocalyx, or E. sessilifolium. In certain
embodiments, the recovery step further comprises obtaining an
extract of the transgenic plant or harvesting material from at
least a portion of the transgenic plant such as an edible portion
of the plant.
[0056] The method of producing a recombinant protein in a
transgenic plant, in certain embodiments may include using an
Agrobacterium system, as described herein, to transform the plant
cell or tissue.
[0057] Preferably, the recombinant proteins produced by this method
are suitable for use as pharmaceuticals, including, but not limited
to, antigens; microbicides; antibodies; hormones; enzymes; blood
components, including, but not limited to, coagulation factors;
interferons, and anticoagulants.
[0058] In certain embodiments, the method involves producing an
antigen protein, preferably a viral protein, more preferably an
avian influenza HA1 antigen.
[0059] In other embodiments, the method involves producing a
microbicide, preferably an antiretroviral microbicide, more
preferably an HIV entry inhibitor, even more preferably
griffithsin.
[0060] The present invention, in certain aspects, also provides
methods of delivering a recombinant protein to a subject comprising
providing harvested material from a transgenic plant of the genus
Eriodictyon that expresses a recombinant protein; and administering
the harvested material to the subject in an amount necessary to
deliver an effective amount of the recombinant protein.
[0061] In certain embodiments, the method of administration
comprises delivery to the subject of an edible portion of the
transgenic plant expressing a recombinant protein. In other
embodiments, the method of administration may comprise mucosal
delivery to the subject of recombinant protein or material
containing such protein harvested from a transgenic plant produced
according to an aspect of the invention.
[0062] As used herein, the term "subject" is used to mean an
animal, preferably a mammal, including a human. The terms "patient"
and "subject" may be used interchangeably. Thus, certain
embodiments of the invention are directed to appropriate dosage
forms useful in the administration of active pharmaceutical
ingredients to a subject.
[0063] As used herein, "mucosal surface" includes, without
limitation, nasal, oral, lingual, sub-lingual, buccal, gingival,
palatal, vaginal, ocular, auditory, pulmonary tract, urethral, and
rectal surfaces. Certain embodiments of the invention relate to
compositions and methods for administration of pharmaceutical
agents to a mucosal surface in a subject.
[0064] A composition comprising a recombinant protein produced
according to an aspect of the invention may be applied to any
mucosal surface as deemed appropriate for the delivery thereof,
including vaginal, rectal, and ocular surfaces. In certain
preferred embodiments, the method of delivery the mucosal surface
is to an intranasal or oral surface of the subject. Preferably, the
oral surface may include, but is not limited to, lingual,
sub-lingual, buccal, gingival, and palatal surfaces.
[0065] In certain preferred embodiments, the recombinant protein
comprises an antigen, preferably a viral antigen, more preferably
an avian influenza HA1 antigen. In this embodiment, the harvested
material which contains the expressed antigen is administered in an
amount sufficient to induce an immune response in the subject. The
immune response may include the induction of cytotoxic T
lymphocytes or the generation of antibodies. Preferably, the
delivery of the antigen to the subject will produce a sufficient
immune response to confer resistance to infection upon the subject.
In any event, the method may be used to generate antibodies to the
antigen which may be used to aid in the purification of the
antigen. Additionally, any generated antibodies may be useful in
the detection of the virus from which the antigen is derived.
[0066] In other embodiments, the recombinant protein is a
microbicide, preferably an antiretroviral microbicide, more
preferably an HIV entry inhibitor, even more preferably
griffithsin. In this embodiment, the harvested material which
contains the expressed microbicide is administered in an amount
sufficient to provide a prophylactic effect. For example, harvested
material from Yerba Santa expressing the microbicide griffithsin
may be administered to the mucosal surfaces (such as the vaginal or
rectal mucosa) of subjects to protect these surfaces from HIV
transmission.
[0067] The present invention, in certain aspects, also provides
methods of propagating a plant in vitro, wherein the plant is
preferably a member of the genus Eriodictyon, the method comprising
the steps of: excising a stem segment, preferably a segment having
a node; and incubating the segment in a growth medium, preferably
MS medium, the medium preferably comprising a cytokinin, more
preferably zeatin; whereby the segment produces a shoot.
Preferably, the plant so propagated is E. californicum, E.
trichocalyx, or E. sessilifolium.
[0068] As used herein, "cytokinins" refer to a class of plant
hormones that promote cell division. Examples of cytokinins include
the adenine-type cytokinins, kinetin, zeatin, benzylaminopurine
(BAP); and the phenylurea-type cytokinins, diphenylurea and
thidiazuron.
[0069] In certain embodiments, the methods may further comprise the
steps of: excising a shoot; and, preferably, incubating the excised
shoot in medium, preferably MS medium, the medium preferably
comprising an auxin, more preferably indole-3-butyric acid; whereby
the shoot produces a root.
[0070] As used herein, "auxins" refer to a class of plant hormones
that control cell expansion. Examples of auxins include the
naturally occurring auxins, 4-chloro-indoleacetic acid,
phenylacetic acid (PAA), and indole-3-butyric acid (IBA),
indole-3-acetic acid (IAA); and the synthetic auxins,
naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid
(2,4-D).
[0071] The methods of the invention may further comprise the steps
of: incubating the shoot, preferably for at least three weeks, in
culture medium, whereby the shoot produces a leaf. The methods may
further comprise the steps of: cutting a segment from the leaf;
placing the segment in a culture medium; and incubating the segment
in the dark, whereby the segment develops callus tissue.
Preferably, the medium is MS medium. Preferably, the medium at
least one of BAP, NAA and 2,4-D. More preferably, the medium
comprises each of BAP, NAA, and 2,4-D.
[0072] In certain aspects, the invention relates to methods for
rapid mass propagation of three Yerba Santa species in vitro, root
induction, induction of cell suspensions, regeneration,
transformation and transfer of plants to greenhouse conditions.
Additionally, methods for the production of callus tissues for all
three species and for the establishment of tissue culture,
including fast growing cell suspensions, are provided. Rapid and
high frequency regeneration from leaf explants is efficient for
these three Yerba Santa species. In certain embodiments, an
efficient transformation protocol is provided and used for the
production of an avian flu antigen or griffithsin in Yerba Santa
leaf tissues. Overall these results provide additional
opportunities to utilize and expand on the beneficial properties of
this unique medicinal herb.
[0073] In certain aspects, the invention relates to methods of
producing a cell suspension culture of a plant, preferably a plant
that is a member of the genus Eriodictyon, the method comprising
the steps of: excising a portion of the callus tissue produced
according to certain aspects of the invention; placing the callus
tissue in a liquid medium, preferably MS medium, the medium
preferably comprising 2,4-D; and incubating the suspension in the
dark with agitation.
[0074] In certain embodiments, the invention relates to protocols
for in vitro propagation, callus induction, shoot regeneration,
establishment of a rapid growing cell suspension culture, and
propagation in greenhouse conditions for Yerba Santa. Rapid and
high frequency regeneration from leaf explants (via shoot
organogenesis) was efficient for all tested Yerba Santa species. An
Agrobacterium --mediated transformation protocol was established.
In certain aspects, transformation conditions are provided for
successful production of transgenic Yerba Santa, including, for
example, the use of a precultivation period, low concentration of
Agrobacterium inoculums, and a multi-step selection procedure.
[0075] Yerba Santa species demonstrated several challenges in
tissue culture and transformation experiments including browning of
explants on different media, difficulties with root induction, high
levels of necrosis after co-cultivation with Agrobacterium and
selection procedures. Some of these difficulties are often
encountered for other medicinal plants. In certain embodiments of
the invention, efficient tissue culture and transformation
protocols for Yerba Santa are provided which may potentially find
use with other recalcitrant medicinal plants.
[0076] Certain aspects of the invention relate to rapid mass
propagation of three species of Yerba Santa. In vitro propagation
may permit the production of pathogen-free material. Propagation
from nodal stem segments may yield plants that are genetically
identical with the donor plants. In certain aspects, techniques
described herein may provide methods for rapid propagation on a
commercial scale of these medicinally important plant species.
[0077] According to particular aspects of the invention, Yerba
Santa cell suspensions are provided, which are demonstrated to be
very fast growing and not to contain large aggregates. This
overcomes some difficulties that may be encountered in developing
and maintaining an efficient cell suspension from different plant
species; including a slow rate of cell growth and the formation of
large clumps during the culture period.
[0078] These plant-cell--suspension cultures may be used for the
production of recombinant proteins, including, but not limited to,
vaccines (Fischer et al., (1999) Journal of Immunol Methods
226:1-10); in certain embodiments, this production may be performed
under controlled certified conditions. The Yerba Santa suspensions
described herein have very good growth rates, comparable to other
fast-growing suspensions described in the relevant literature.
[0079] The following examples are provided for the purpose of
further illustrating the present invention but are by no means
intended to limit the same.
EXAMPLES
Materials and Methods
[0080] Plant Material and Propagation In Vitro
[0081] Plant material of three Yerba Santa species (Eriodictyon
californicum, Eriodictyon sessilifolium, and Eriodictyon
trichocalyx) are obtained from Rancho Santa Ana Botanic Garden
(Claremont, Calif.) and Las Pilitas Nursery (Santa Margarita,
Calif.). Segments of stem are excised from Yerba Santa plants,
washed thoroughly under running tap water, and then are dipped in
70% ethanol for 1 min, followed by a 25 min soak in a 1.2% solution
of commercial sodium hypochlorite. After rinsing 3 times with
sterile distilled water, segments with 1 or 2 nodes are transferred
to Phytatrays containing MSP media. Phytatrays are sealed with
Parafilm and incubated in a growth chamber at 24.degree. C. at 16
h-light/8 h-dark photoperiods with light intensity of 40 uE/m2/S1.
In vitro cultures are maintained by transferring 1-cm-long shoot
segments at 5-6 week intervals onto fresh medium. For root
induction 3-4 cm shoots are placed in root induction media MSRI
(Table 1). Rooted plants are transferred to pods containing soil
Metromix and sand (3:1).
TABLE-US-00001 TABLE 1 Media for tissue culture and transformation
experiments Name Media composition MS0 Basic MS basal medium with
3% sucrose, 0.8% agar MSP MS0 with 1 mg/l zeatin MSC-1 MS0 with 0.3
mg/l BAP, 0.5 mg/l NAA, 1 mg/l 2,4-D MSC-2 MS0 with 1 mg/l 2,4-D
MSS MS with 3% sucrose, 0.5 mg/l 2,4-D (liquid) MSR MS with 3%
sucrose, 1 mg/l zeatin, 0.1 mg/l NAA, 5 mg/l silver nitrate 0.9%
agar MSRI MS0 with 1 mg/l lBA MST-1 MS0 with 100 .mu.M
acetosyringone MST-2 MSR with 300 mg/l timentin MST-3 MSR with 50
mg/l kanamycin, 300 mg/l timentin MST-4 MSR with 70 mg/l kanamycin,
250 mg/l timentin MST-5 MSR with 100 mg/l kanamycin, 200 mg/l
timentin MST-6 MS0 with 1 mg/l lBA, 150 mg/l timentin
[0082] Callus Initiation, Cell Suspensions
[0083] Leaf segments (0.5-0.7 cm) are cut from the 3-4 week-old in
vitro propagating shoots from three Yerba Santa species and placed
in Petri dishes (100.times.15 mm) on MSC-1 medium for induction of
callus. Plates are incubated in the darkness at 24.degree. C. for
4-6 weeks. Well developed calli are selected and transferred to
MSC-2 medium. Callus tissue is maintained on MSC-2 medium at 3- to
4-week intervals. All the plates with callus cultures are incubated
in the dark at 24-25.degree. C. Friable callus is used for
initiation of cell suspensions. Approximately 1 g fresh weight of
callus tissue are transferred into 50 ml of medium MSS medium in
sterile 250 ml conical flasks. Cell cultures are grown on a rotary
shaker at 130 rpm in the dark at 25.degree. C. In order to maintain
suspension culture a portion (2-3 ml) of liquid suspension cells
are transferred to fresh MSS medium at 10-14 day-intervals.
[0084] Generation of Transgenic Plants
[0085] Leaf segments (0.5-0.7 cm) are cut from the 3-4 week-old in
vitro propagating shoots of three Yerba Santa species and placed in
Petri dishes (100.times.15 mm) on MSR medium. Ten to twelve
explants per Petri dish are cultivated for 6 weeks and tested for
shoot regeneration efficiency. Agrobacterium tumefaciens strain LBA
4404 is grown overnight in LB medium supplemented with appropriate
antibiotics at 28.degree. C. Binary vector pBIN-Plus (ImpactVector,
Wageningen, the Netherlands) harboring an expression cassette of
avian flu HA1 antigen fused with Fc and driven by the Rubisco
promoter is used. The expression cassette contains the c-Myc and
His6 tags at the C-terminus. The vector also contains the npt II
gene for kanamycin selection of transgenic plants. Leaf segments
are inoculated with Agrobacterium suspension (OD.sub.600 0.5, 0.3,
0.1 or 0.03) for 10 min. After blotting dry with sterile filter
paper, explants are transferred to MST-1 co-cultivation medium
supplemented with acetosyringone (Table 1) and incubated in the
dark for 2 or 3 days at 24.degree. C. To determine the effect of
preculture on transformation efficiency, explants are cultured for
2, 3 or 4 days on MSC-1 medium before inoculation with
Agrobacterium. After co-cultivation, explants are transferred to
MST-2 medium without selection for 7, 10 and 14 days and then
transferred to MST-3 regeneration selection medium with 50 mg/l
kanamycin. After 3 weeks, regenerated green shoots are transferred
to second selection medium MST-4 with increased concentration of
kanamycin (70 mg/l), and after an additional 3 weeks explants are
transferred to third selection medium MST-5 with high kanamycin
concentration (100 mg/l). Putative transgenic shoots are excised
and transferred to rooting medium (MSRI supplemented with 150 mg/l
timentin). Plantlets with roots are transferred to pots containing
a mixture of soil and sand.
Example 1
In Vitro Propagation and Root Induction
[0086] For establishment of in vitro cultures stem segments with
nodes of three Yerba Santa species (Eriodictyon californicum,
Eriodictyon sessilifolium, and Eriodictyon trichocalyx) are used as
primary explants. They are washed, surface sterilized and placed on
MS medium (Murashige T& Skoog F.(1962) Physiol Plant
15:473-497) supplemented with different cytokinins:
N.sup.6-benzylaminopurine (BAP), zeatin, and kinetin. All three
Yerba Santa species propagate in vitro most efficiently on basal MS
medium supplemented with 1 mg/l zeatin. During 6 weeks a
significant number of shoots are produced for all three species.
Comparison of the shoot propagation capacity of three species of
Yerba Santa demonstrates the highest efficiency in E. trichocalyx
(FIG. 1, A and B), about 8-10 shoots are produced from each explant
in 4-5 weeks. A single explant of this species can produce
thousands of shoots in 3-4 months.
[0087] Individual shoots are excised and transferred to
hormone-free medium for rooting. Yerba Santa species demonstrate
different root formation capacities. E. sessilifolium start to form
roots after 7-10 days on media without hormones (FIG. 1C), whereas
E. californicum and E. trichocalyx show significant difficulties
with root formation. Therefore media with different auxins are
tested for stimulation of root formation: naphthalene acetic acid
(NAA), indole-3-acetic acid (IAA), and indole-3-butyric acid (IBA).
The best root development for both cultivars is recorded on medium
supplemented with 1 mg/l IBA (FIG. 1, D and E), however some of the
shoots are not able to produce roots. After testing shoots of
different size and development, it appears that large (3-4 cm),
healthy and well developed shoots of E. californicum and E.
trichocalyx are capable of producing roots. Rooted plants (FIG. 1F)
are transferred to pods with mixture of soil (Metromix) and sand
(3:1) (FIGS. 1G and 1H). Morphology of the micropropagated plants
are identical to that of normal propagated plants.
Example 2
Establishment of Cell Suspensions
[0088] To obtain a rapidly growing cell suspension culture, three
Yerba Santa species are screened for callus induction and
cultivation. Callus tissues are initiated for all three Yerba Santa
species from leaf explants on callus induction media MSC-1 after
4-6 weeks of incubation in darkness. For future propagation, callus
tissues are transferred to MSC-2 medium. After 6-8 weeks of
cultivation on MSC-2 medium, the callus of E. trichocalyx showed
the best growth capacities (FIG. 2A) E. californicum also
demonstrated good growth capacity, however some part of the callus
tissues showed browning (FIG. 2B).
[0089] Cell suspensions of Yerba Santa species are established from
callus tissues in liquid media MSS. E. trichocalyx show the best
results, including fast growth rates of cells, no browning of
cells, and mild cell aggregations (FIGS. 2C and 2D). The E.
trichocalyx cell suspension is very fast growing: about a
10-12-fold increase in cell volume is achieved in 5-7 days. (FIG.
3). These results are comparable with most fast growing cell
suspension of other plant species (Fischer R & Schillberg S
(eds) (2004) Molecular Farming. Plant-made Pharmaceuticals and
Technical Proteins Wiley-VCH Verlag GmbH & Co. Weinbeim) and
can provide a very efficient system for the production of vaccines
and other recombinant proteins.
Example 3
Transformation of Yerba Santa
[0090] As a first step in the development of an efficient
transformation system, the development of an efficient regeneration
system is initiated. Preliminary experiments using different media
compositions and types of explants indicate that shoots from
several Yerba Santa species regenerate most efficiently on MSR
medium (Table 1) and that leaf segments have the best regeneration
potential. During 5-6 weeks leaf explants produce multiple shoots
on MSR medium through direct regeneration without callus formation.
Comparison of the regeneration capacity of three species (E.
californicum, E. sessilifolium, and E. trichocalyx) reveal the
highest regeneration efficiency in E. trichocalyx leaf segments
reaching 75%-82% (FIG. 4; FIGS. 5A and 5B). Based on the results of
regeneration experiments, leaf segments of E. trichocalyx are
chosen as explants for transformation experiments.
[0091] Preliminary transformation experiments reveal several
challenges associated with inoculation and selection for Yerba
Santa species. In the first series of experiments, the efficiency
of the inoculation procedure is tested. The exposure of leaf
explants to Agrobacterium culture at OD.sub.600 0.5 cause severe
necrosis in most of the treated Yerba Santa explants.
Pre-cultivation of E. trichocalyx leaf explants is tested for 2, 3
and 5 days before inoculation with Agrobacterium OD.sub.600 0.5.
However, this approach does not appear to decrease the number of
browning tissues after inoculation and co-cultivation procedures.
In an effort to reduce necrosis of explants in response to
Agrobacteria, the Agrobacteria suspension is diluted to OD.sub.600
0.3, 0.1, and then finally to 0.03. Inoculation of leaf segments
with a suspension diluted to 0.03 significantly decreases the level
of necrosis. In the same set of experiments, it is demonstrated
that 2 days of co-cultivation significantly decreased necrosis, as
compared to 3 days. The addition of polyvinylpyrrolidone (250 mg/l)
and increasing the agar concentration in the media also prove to be
beneficial.
[0092] Several selection schemas are tested. No transgenic plants
are recovered when selection is started immediately after
co-cultivation. Therefore, the effect of a delay period is tested,
during which explants are kept in MST-2 non-selection medium
supplemented with timentin for Agrobacterium elimination for 7, 10
or 14 days. Transgenic shoot regeneration from explants is highest
when leaf explants are left on MST-2 medium for 10 days, then
explants are transferred to selection medium. Among different
selection systems tested the best results are obtained with a
3-step selection procedure with a gradually increasing
concentration of kanamycin in the selection regeneration media. Use
of a relatively low concentration of kanamycin (50 mg/l) in the
first selection media reveals good survival of tissues and a large
percent of escapes. After 3 weeks, the explants are transferred to
a second selection medium containing 70 mg/l kanamycin and finally
to a selection medium with 100 mg/l kanamycin (FIG. 5C). When
shoots reach 3-4 cm in length (FIG. 5D), they are transferred to
root induction media MSRI with addition of 150 mg/l timentin (FIG.
6A). Rooted plants are transferred to pods with mixture of soil and
sand (FIG. 6B).
Example 4
Expression of Avian Influenza Antigen
[0093] Leaf tissues of transgenic plants are tested for level of
expression of avian influenza antigen. Western blot analysis with
C-myc antibodies reveals a protein band of the expected molecular
size in the leaf tissue of transgenic plants (FIG. 7). The
morphology of the transgenic plants is identical to that of
non-transgenic plants (FIG. 6B and FIG. 1H).
Example 5
Immunological Assessment of Plant-Expressed Avian Influenza Antigen
in Mice
[0094] Groups of 6- to 8-week-old female BALB/c mice (five mice per
group) are used in all experiments. The experiment is performed
using Eriodictyon californicum, Eriodictyon sessilifolium, or
Eriodictyon trichocalyx plants that have been transformed to
express HA1 antigen and wild type (WT) plants.
[0095] For oral immunization experiments, groups of 6- to
8-week-old female BALB/c mice (five mice per group) are used. The
experiment are performed using Yerba Santa plants that express HA1
antigen and wild type (WT) plants. Each mouse is fed with 2-3 g of
fresh leaf tissue over a period of 6-8 h. Control mice receive wild
type plant material. Mice are immunized 3 times at 2-week
intervals.
[0096] For intranasal immunization experiments, groups of 6- to
8-week-old BALB/c mice (five per group) are used. 2 .mu.g of Yerba
Santa-derived antigen is administered in 10 .mu.l of saline into
both nostrils (5 .mu.l in each). In some groups, plant material is
supplemented with 1 .mu.g of CT (cholera toxin) as an adjuvant.
Control mice receive wild type plant material. Mice are immunized 3
times at 2-week intervals.
[0097] Blood and fecal matter is collected 10 days after each
immunization. Protein from fecal pellets will be extracted in PBS
(10 vol/wt) supplemented with 1% BSA and protease inhibitors. Mice
are killed 10 days after the last immunization and bled by cardiac
puncture. Sera and fecal pellets are analyzed for the presence of
antigen (HA1) specific antibodies by Western blot analysis and
ELISA.
[0098] Solid-phase ELISA is carried out as described in Hooper et
al. (2001) J. Immunol. 167, 3470-3477 MaxiSorp 96-well plates
(Nalge Nunc) are coated overnight at 4.degree. C. with the HA1 at a
concentration of 1 .mu.g/ml in PBS. Extract are diluted initially
1:10 in PBS and diluted serially 1:2 in the same buffer incubation
for 1 hour at 37.degree. C. Antigen-specific antibodies are
detected by using the following antibodies: rabbit anti-mouse IgG
(total) and anti-mouse IgG1 (both from BD Biosciences Pharmingen),
anti-mouse IgG2a, IgG2b, IgG3 and IgA (all from Organon Teknika),
and anti-mouse IgE (eBioscience, San Diego) HRP-conjugated (diluted
1:2000 in PBST) for 1 h at 37 C. Between each step, wells are
washed four times with PBST. Finally, plates are developed in a
solution of OPD peroxidase substrate (Sigma Chemical). Absorbance
at 490 nm is determined using a microplate reader.
[0099] The foregoing examples and description of the preferred
embodiments should be taken as illustrating, rather than as
limiting the present invention as defined by the claims. As will be
readily appreciated, numerous variations and combinations of the
features set forth above can be utilized without departing from the
present invention as set forth in the claims. Such variations are
not regarded as a departure from the spirit and scope of the
invention, and all such variations are intended to be included
within the scope of the following claims.
[0100] In addition, where features or aspects of the invention are
described in terms of Markush group or other grouping of
alternatives, those skilled in the art will recognized that the
invention is also thereby described in terms of any individual
member or subgroup of members of the Markush group or other
group.
[0101] Unless indicated to the contrary, all numerical ranges
described herein include all combinations and subcombinations of
ranges and specific integers encompassed therein. Such ranges are
also within the scope of the described invention.
[0102] The disclosures of each patent, patent application and
publication cited or described in this document are hereby
incorporated herein by reference, in their entirety.
* * * * *