U.S. patent application number 12/150900 was filed with the patent office on 2009-01-15 for vectors and cells for preparing immunoprotective compositions derived from transgenic plants.
Invention is credited to Guy A. Cardineau, Dwayne D. Kirk, Hugh Stanley Mason, Joyce M. VanEck, Amanda Maree Walmsley.
Application Number | 20090017065 12/150900 |
Document ID | / |
Family ID | 33435152 |
Filed Date | 2009-01-15 |
United States Patent
Application |
20090017065 |
Kind Code |
A1 |
Cardineau; Guy A. ; et
al. |
January 15, 2009 |
Vectors and cells for preparing immunoprotective compositions
derived from transgenic plants
Abstract
The inventions is drawn towards vectors and methods useful for
preparing genetically transformed plant cells that express
immunogens from pathogenic organisms which are used to produce
immunoprotective particles useful in vaccine preparations. The
invention includes plant optimized genes that encode the HN protein
of Newcastle Disease Virus. The invention also relates to methods
of producing an antigen in a transgenic plant.
Inventors: |
Cardineau; Guy A.; (Tempe,
AZ) ; Mason; Hugh Stanley; (Phoenix, AZ) ;
VanEck; Joyce M.; (Ithaca, NY) ; Kirk; Dwayne D.;
(Mesa, AZ) ; Walmsley; Amanda Maree; (Mesa,
AZ) |
Correspondence
Address: |
Edwards Angell Palmer & Dodge LLP
P.O. Box 55874
Boston
MA
02205
US
|
Family ID: |
33435152 |
Appl. No.: |
12/150900 |
Filed: |
April 30, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11450102 |
Jun 8, 2006 |
7407802 |
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12150900 |
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10838834 |
May 4, 2004 |
7132291 |
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11450102 |
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60467998 |
May 5, 2003 |
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Current U.S.
Class: |
424/206.1 ;
424/214.1; 800/288; 800/298 |
Current CPC
Class: |
A61P 37/04 20180101;
A61P 31/14 20180101; C12N 15/8258 20130101 |
Class at
Publication: |
424/206.1 ;
800/298; 424/214.1; 800/288 |
International
Class: |
A61K 39/145 20060101
A61K039/145; A01H 5/00 20060101 A01H005/00; C12N 15/82 20060101
C12N015/82; A61P 37/04 20060101 A61P037/04; A61K 39/17 20060101
A61K039/17 |
Claims
1. A transgenic plant comprising a vector, wherein said vector
comprises SEQ ID NO: 1.
2. A transgenic plant comprising a vector, wherein said vector is
selected from the group consisting of pCHN, pGHN, pGHN151, pGHN153,
pMHN, pUHN.
3. The transgenic plant of claim 1, wherein a plant-functional
promoter is operably linked to SEQ ID NO:1.
4. A vaccine comprising a recombinant viral antigenic protein and a
pharmaceutically acceptable carrier, wherein the viral antigenic
protein is the HN antigen of Newcastle Disease virus produced by a
vector comprising SEQ ID NO: 1 or a vector selected from the group
consisting of pCHN, pGHN, pGHN151, pGHN153, pMHN, pUHN, and wherein
said vaccine is capable of eliciting an immune response upon
administration to an animal.
5. The vaccine of claim 4, wherein said HN protein comprises SEQ ID
NO:2.
6. The vaccine of claim 4, wherein said HN protein is produced in a
plant cell.
7. A method for protecting an animal against NewCastle Disease
Virus comprising administering an effective amount of the vaccine
of claim 4 to an animal.
8. A vaccine comprising a recombinant viral antigenic protein and a
pharmaceutically acceptable carrier, wherein the viral antigenic
protein is the HA antigen of Avian Influenza Virus produced by a
vector for expressing an immunoprotective antigen in a plant cell
comprising a DNA sequence encoding the HA antigen of Avian
Influenza Virus, wherein the vector is pCHA, and wherein said
vaccine is capable of eliciting an immune response upon
administration to an animal.
9. The vaccine of claim 8, wherein said HA antigen is produced in a
plant cell.
10. A method for protecting an animal against Avian Influenza Virus
comprising administering an effective amount of the vaccine of
claim 8 to an animal.
11. The method of claim 7 or 10, wherein the vaccine is
administered orally, intranasaly, intraperitonealy,
intramuscularly, intravenously or subcutaneously.
12. The method of claim 7 or 10, wherein the effective amount is at
a range of 1 .mu.g to 50 .mu.g per kilogram of bodyweight.
13. A method of producing an antigen in a transgenic plant
comprising the steps of: a) producing a transgenic plant comprising
a vector encoding said antigen; b) incubating said plant under
conditions wherein said plant expresses said antigen; and wherein
said plant is incubated prior to the onset of ripening.
14. The method of claim 13, wherein said plant comprises a fruit
that ripens.
15. The method of claim 13, wherein said plant is a tomato
plant.
16. The method of claim 14, wherein the fruit of said plant is
harvested prior to the onset of ripening.
17. The method of claim 16, wherein said antigen is isolated from
said harvested fruit.
18. The method of claim 13, wherein said antigen is selected from
the group consisting of HN antigen of Newcastle Disease Virus, HA
antigen of Avian Influenza Virus, LTB, NVCP, zona pellucida
glycoprotein and HBsAg.
Description
[0001] This application is a divisional of Ser. No. 11/450,102,
filed Jun. 8, 2006, which is a divisional of U.S. Pat. No.
7,132,291, filed May 4, 2004, which claims the benefit of U.S.
Provisional Application No. 60/467,998, filed on May 5, 2003. The
entire teachings of the above applications are incorporated herein
by reference.
FIELD OF INVENTION
[0002] The present invention generally relates to the field of
plant molecular biology as it applies to the recombinant production
of plant-made vaccines.
BACKGROUND OF THE INVENTION
[0003] Recombinant DNA technology has provided substantial
improvements in the safety, quality, efficacy and cost of
pharmaceutical and veterinary medicaments including vaccines. Plant
produced mucosal vaccines were invented by Curtiss & Cardineau.
See U.S. Pat. Nos. 5,654,184; 5,679,880 and 5,686,079 herein
incorporated by reference. Others have described transgenic plants
expressing immunoprotective antigens and methods for production
including Arntzen, Mason and Lam. See U.S. Pat. Nos. 5,484,717;
5,914,123; 6,034,298; 6,136,320; 6,194,560; and 6,395,964 herein
incorporated by reference.
[0004] Vaccines produced in plant systems offer a number of
advantages over conventional production systems. Conventionally
produced vaccines strains (live and vectored) may revert towards
virulence or carry biological contaminants from the production
process. Subunit vaccines may be difficult to produce and purify
due to protein instability issues and will not be glycosylated when
produced in prokaryotes.
[0005] Plant cell production avoids the need for animal-sourced
components in growth media essentially eliminating the risk of
transmitting pathogenic contaminants from the production process.
Plant cells are capable of post translational glycosylation, and
plant cell growth media is generally less expensive and easier to
handle and prepare compared to conventional growth media presently
used in the manufacture of vaccines.
[0006] Systemic immunity to a particular pathogen results from
activation of the immune system in response to antigen presented by
a particular pathogenic organism or via a vaccine designed to
protect against a particular pathogenic agent. Exposure to a
pathogen is often through mucosal surfaces that are constantly
exposed and challenged by pathogenic organisms.
[0007] Mucosal and oral immunity results from the production of
sIgA (secretory IgA) antibodies in secretions that bathe all
mucosal surfaces of the respiratory tract, gastrointestinal tract
and the genitourinary tract and in secretions from all secretory
glands. McGhee, J. R. et al., Annals N Y. Acad. Sci. 409, (1983).
These sIgA antibodies act to prevent colonization of pathogens on a
mucosal surface (Williams, R. C. et al., Science 177, 697 (1972);
McNabb, P. C. et al., Ann. Rev. Microbiol. 35, 477 (1981) and thus
act as a first line of defense to prevent colonization or invasion
through a mucosal surface. The production of sIgA can be stimulated
either by local immunization of the secretory gland or tissue or by
presentation of an antigen to either the GALT (gut-associated
lymphoid tissue or Peyer's patches) or the BALT
(bronchial-associated lymphoid tissue). Cebra, J. J. et al., Cold
Spring Harbor Symp. Quant. Biol. 41, 210 (1976); Bienenstock, J.
M., Adv. Exp. Med. Biol. 107, 53 (1978); Weisz-Carrington, P. Et
al., J. Immunol. 123, 1705 (1979); McCaughan, G. et al., Internal
Rev. Physiol 28, 131 (1983). Membranous microfold cells, otherwise
known as M Cells, cover the surface of the GALT and BALT and may be
associated with other secretory mucosal surfaces. M cells act to
sample antigens from the luminal space adjacent to the mucosal
surface and transfer such antigens to antigen-presenting cells
(dendritic cells and macrophages), which in turn present the
antigen to a T lymphocyte (in the case of T-dependent antigens),
which process the antigen for presentation to a committed B cell. B
cells are then stimulated to proliferate, migrate and ultimately be
transformed into an antibody-secreting plasma cell producing IgA
against the presented antigen. When the antigen is taken up by M
cells overlying the GALT and BALT, a generalized mucosal immunity
results with sIgA against the antigen being produced by all
secretory tissues in the body. Cebra et al., supra; Bienenstock et
al., supra; Weinz-Carrington et al., supra; McCaughan et al.,
supra. Oral immunization is therefore a most important route to
stimulate a generalized mucosal immune response and, in addition,
leads to local stimulation of a secretory immune response in the
oral cavity and in the gastrointestinal tract.
[0008] Mucosal immunity can also be advantageously transferred to
offspring. Immunity in neonates may be passively acquired through
colostrum and/or milk. This has been referred to as lactogenic
immunity and is an efficient way to protect animals during early
life. sIgA is the major immunoglobulin in milk and is most
efficiently induced by mucosal immunization.
[0009] The M cells overlying the Peyer's patches of the
gut-associated lymphoid tissue are capable of taking up a diversity
of antigenic material and particles (Sneller, M. C. and Strober,
W., J. Inf. Dis. 154, 737 (1986). Because of their abilities to
take up latex and polystyrene spheres, charcoal, microcapsules and
other soluble and particulate matter, it is possible to deliver a
diversity of materials to the GALT independent of any specific
adhesive-type property of the material to be delivered.
[0010] Vectors and cells useful for producing transgenic
plant-derived immunoprotective antigens, and improved methods of
antigen production would greatly facilitate the development,
manufacture and efficacy plant-produced vaccines.
SUMMARY OF THE INVENTION
[0011] The invention is based on plant optimized sequences encoding
an immunoprotective antigen of interest. In particular, the
invention is based on a plant optimized DNA sequence encoding the
HN antigen of Newcastle Disease Virus or a DNA sequence encoding
the HA antigen of Avian Influenza Virus. The invention also
includes a recombinant expression vector for effecting expression
of an immunoprotective antigen gene in a plant cell, as well as
plant cells and transgenic plants comprising the expression vector,
as well as vaccines comprising a protein product of the expression
vector. The invention also relates to methods of protecting against
the effects of a pathogen utilizing the vaccines of the invention.
The invention further relates to methods of producing an antigen in
a transgenic plant.
[0012] The invention provides for an isolated plant optimized
nucleotide sequence encoding the HN antigen of Newcastle Disease
Virus comprising the sequence of SEQ ID NO: 1, as well as a
recombinant expression vector comprising SEQ ID NO: 1.
[0013] In one embodiment, the vector is selected from the group
consisting of pCHN, pGHN, pGHN151, pGHN153, pMHN, pUHN.
[0014] In another embodiment, the vector comprises a
plant-functional promoter is operably linked to SEQ ID NO: 1.
[0015] The invention also provides for a recombinant expression
vector for expressing an immunoprotective antigen in a plant cell
comprising a DNA sequence encoding the HA antigen of Avian
Influenza Virus, wherein the vector is pCHA
[0016] The invention further provides for a transgenic plant cell
for expression of an immunogenic antigen comprising a vector of the
invention. The plant cell includes a tomato plant cell or a tobacco
plant cell, as well as a cell from any of the plant species
described hereinbelow.
[0017] The invention further provides for a transgenic plant
comprising a vector of the invention.
[0018] The invention also provides for a vaccine comprising a
recombinant viral antigenic protein and a pharmaceutically
acceptable carrier, wherein the viral antigenic protein is the HN
antigen of Newcastle Disease virus produced by a vector of the
invention, and wherein the vaccine is capable of eliciting an
immune response upon administration to an animal.
[0019] In one embodiment, the HN protein of the vaccine comprises
SEQ ID NO:2. The HN protein of the vaccine can be produced in a
plant cell.
[0020] The invention also provides for a vaccine comprising a
recombinant viral antigenic protein and a pharmaceutically
acceptable carrier, wherein the viral antigenic protein is the HA
antigen of Avian Influenza Virus produced by a vector of the
invention, and wherein the vaccine is capable of eliciting an
immune response upon administration to an animal. In one
embodiment, the HA antigen of the vaccine is produced in a plant
cell.
[0021] The invention also provides for a method for protecting an
animal against NewCastle Disease Virus or Avian Influenza Virus
comprising administering an effective amount of the appropriate
vaccine of the invention to an animal. According to one embodiment
of the method, wherein the vaccine is administered orally,
intranasaly, intraperitonealy, intramuscularly, intravenously or
subcutaneously. In one embodiment of the method, the effective
amount of the vaccine is at a range of 1 .mu.g to 50 .mu.g per
kilogram of body weight.
[0022] The invention also provides for a method of producing an
antigen in a transgenic plant comprising the steps of: a) producing
a transgenic plant comprising a vector encoding the antigen; b)
incubating the plant under conditions wherein the plant expresses
the antigen; and wherein the plant is incubated prior to the onset
of ripening.
[0023] In one embodiment, the plant comprises a fruit that
ripens.
[0024] In another embodiment, the plant is a tomato plant.
[0025] In another embodiment, the fruit of the plant is harvested
prior to the onset of ripening. According to one embodiment of this
method, the antigen is isolated from the harvested fruit.
[0026] In another embodiment, the antigen is selected from the
group consisting of HN antigen of Newcastle Disease Virus, HA
antigen of Avian Influenza Virus, LTB, NVCP, zona pellucida
glycoprotein and HBsAg.
BRIEF DESCRIPTION OF THE FIGURES
[0027] FIGS. 1a and 1b. The plant optimized coding sequence (SEQ ID
NO: 1) and protein sequence (SEQ ID NO: 2) of the HN gene of NDV
strain "Lasota"
[0028] FIG. 2. Map of pBBV-PHAS-iaaH that contains the plant
selectable marker PAT (phosphinothricin acetyl transferase),
includes the constitutive CsVMV (cassaya vein mosaic virus)
promoter and is terminated by the MAS 3' (mannopine synthase)
element. LB and RB (left and right T-DNA border) elements from
Agrobacterium delineate the boundaries of the DNA that is
integrated into the plant genome.
[0029] FIG. 3. Map of pCP!H which is a "template vector" used as a
starting plasmid for a variety of plant expression vectors for
expressing immunoprotective antigens.
[0030] FIG. 4. Map of pCHN expression vector for NDV HN protein.
This vector comprising the HN expression cassette includes the
constitutive CsVMV promoter and is terminated by the soybean vspB
3' element.
[0031] FIG. 5. Map of pgHN expression vector for NDV HN protein.
This vector comprising the HN expression cassette includes the
tuber-specific GBSS promoter with TEV 5' UTRand is terminated by
the soybean vspB 3' element.
[0032] FIG. 6. Map of pgHN151 expression vector for NDV HN protein.
The HN expression vector or cassette includes the tuber-specific
GBSS promoter with its native 5' UTR and intron, and is terminated
by the soybean vspB 3' element. The vector is derived from
pBBV-PHAS-iaaH, which contains the plant selectable marker PAT,
includes the CsVMV promoter and is terminated by the MAS 3'
element. LB and RB, left and right T-DNA border elements delineate
the boundaries of the DNA that is integrated into the plant
genome.
[0033] FIG. 7. Map of pgHN153 expression vector for NDV HN protein.
The HN expression vector includes the tuber-specific GBSS promoter
with its native 5' UTR and intron, and is terminated by the bean
phaseolin 3' element. The vector is derived from pBBV-PHAS-iaaH,
which contains the plant selectable marker PAT, includes the CsVMV
promoter and is terminated by the MAS 3' element. LB and RB, left
and right T-DNA border elements delineate the boundaries of the DNA
that is integrated into the plant genome.
[0034] FIG. 8. Map of pMHN expression vector for NDV HN protein.
The HN expression vector includes the constitutive 40CSAMAS
promoter (P2 direction) and is terminated by the soybean vspB 3'
element. The vector is derived from pBBV-PHAS-iaaH, which contains
the plant selectable marker PAT, includes the CsVMV promoter and is
terminated by the MAS 3' element. LB and RB, left and right T-DNA
border elements delineate the boundaries of the DNA that is
integrated into the plant genome.
[0035] FIG. 9. Map of pCHA expression vector for the HA gene of the
AIV A/turkey/Wisconsin/68 (H5N9).
[0036] FIG. 10. The DNA (SEQ ID NO: 3) and protein (SEQ ID NO: 4)
sequences of the HA gene of AIV A/turkey/Wisconsin/68 (H5N9).
[0037] FIG. 11. Map of pGLTB intermediate vector.
[0038] FIG. 12. Map of pCLT105 intermediate vector.
[0039] FIG. 13. HA expression in transgenic NT1 cell lines using
pGPTV-HAO or pCHA. Callus cells growing on solid media were
extracted and assayed for HA by ELISA and for total protein by the
Bradford method. Data are presented as ng HA per .mu.g total
protein. A01/12, a high-expressing line selected from several
pGPTV-HAO-transformed lines. CHA, lines transformed with pCHA.
CVMV/13, a vector-only transformed line. Separate samples were
extracted and assayed Jul. 12, 2001 or Jul. 27, 2001.
[0040] FIG. 14. Repeated assays of pCHA-transformed NT1 cell
lines.
[0041] FIG. 15. Western blot for AIV HA expression in
pCHA-transformed NT1 cell lines. NT1 cell lines were grown in
liquid suspension culture, and extracts were resolved by SDS-PAGE,
electro-transferred to PVDF membrane, and probed with chicken
anti-AIV-H5 from USDA/SEPRL, which is also used as the detector
antibody in the HA quantitation ELISA. Lanes 1 and 10, molecular
size standards; lane 2, HN Reference Antigen at 1:800, 31.25
ng/well; lane 3, CHA-13 (1:2); lane 4, CHA-42 (1:2); lane 5, CHA-43
(1:2); lane 6, CHA-44 (1:2); lane 7, CHA-61 (1:2); lane 8, GPTV-HAO
grown with Kanamycin (1:2); lane 9, GPTV-HAO grown without
Kanamycin (1:2).
[0042] FIG. 16. HA expression in microtubers of pCHA-transformed
potato plantlets. Microtubers were generated in vitro from stem
nodes of tissue culture plantlets. Samples were extracted and
assayed for HA by ELISA. Data are presented as ng HA per g fresh
microtuber weight. Line numbers indicate independent transgenic
lines. Desiree, a non-transformed line. Standard error bars
represent standard deviation of multiple determinations.
[0043] FIG. 17. HA expression in leaves of pCHA-transformed potato
plants. Leaf samples from greenhouse-grown plants were extracted
and assayed for HA by ELISA and for total protein by the Bradford
method. Data are presented as ng HA per .mu.g total protein. Line
numbers indicate independent transgenic lines. Standard deviations
of multiple determinations are shown.
[0044] FIG. 18. HA expression in tubers of soil-grown
pCHA-transformed potato plants. Greenhouse-grown plants were
harvested and tubers sampled for extraction and ELISA for HA
expression. Four replicate samples were analyzed, and the standard
deviations are shown. Data are presented as ng HA per g fresh tuber
weight.
[0045] FIG. 19. Expression of NDV-HN in NT1 cells transformed with
pCHN. Extracts f cells growing on solid media were assayed by ELISA
for NDV-HN, using inactivated NDV as a reference standard. Total
soluble protein (TSP) was assayed by the Bradford method (BioRad)
using bovine serum albumin as a standard.
[0046] FIG. 20. Expression of HN per cell mass in pCHN-transformed
NT1 lines.
[0047] FIG. 21. Stability of expression of HN in pCHN-transformed
NT1 cell lines.
[0048] FIG. 22. Western blot of pCHN-transformed NT1 cells using
HN-specific antibodies. NT1 cells cultured on solid media (5P, 7P)
or in liquid suspension (5-6, 5-13, 7-6, 7-13) were extracted and
subjected to SDS-PAGE, followed by Western blot probed with either
monoclonal (left) or polyclonal (right) antibodies. MW, molecular
weight markers, indicated by numbers at left in kDa. Lanes 11.9+,
5.85+, 2.99+, 1.45+ indicate amount (ng) of reference standard
inactivated NDV run in those lanes. C, nontransgenic NT1 cell
extract.
[0049] FIG. 23. HN antigen is maintained in freeze-dried
pCHN-transformed NT1 cells and on storage of extracts at 4.degree.
C. NT1 cells were freeze-dried, extracted, and subjected to ELISA.
The results for freeze-dried cells were analyzed by either a log or
linear regression model, as indicated in the inset. The values are
corrected to indicate HN content per mass of fresh weight cells
using estimates of water loss on drying. Fresh cells were extracted
and analyzed immediately (2/19/2) or stored at 4.degree. C. for 1
week prior to assay.
[0050] FIG. 24. Sucrose gradient analysis of HN antigen shows
particulate character. Extracts of pCHN-transformed NT1 cell lines
CHN-7 or CHN-18, or reference standard inactivated NDV were
sedimented in 10-50% sucrose/PBS gradients at 350,000 g for 5 h.
Fractions were collected and assayed by ELISA for HN. Fraction 1 is
the top of gradient.
[0051] FIG. 25. Expression of HN in pMHN- and pCHN-transformed NT1
cell lines.
[0052] FIG. 26. HN expression in pCHN-transformed potato.
[0053] FIG. 27. Particle behavior of HN antigen extracted from
pCHN-transformed potato tubers.
[0054] FIG. 28. HN expression in microtubers of pGHN-transformed
potato plants.
[0055] FIG. 29. Expression of HN in tubers of pGHN- and
pGHN151-transformed potato plants.
[0056] FIG. 30. T-DNA region from the construct pCHN.
[0057] FIG. 31. Effect of ripening on wild type TA234 tomato fruit
pH.
[0058] FIG. 32. Effect of ripening on wild type TA234 tomato fruit
total soluble protein.
[0059] FIG. 33. Southern analysis of T.sub.0 CHN tomato lines. The
positive control is an EcoRI digest of the plasmid pCHN loaded to
indicate the intensity of 2 copies of the HN gene per genome while
the negative control is DNA from wild type TA234 tomato.
[0060] FIG. 34. Total RNA from wild type and transgenic tomato
fruit. (a) Methylene blue stain of membrane. (b) Northern analysis.
NC represents the negative control, total RNA from wild type fruit;
L, MBI Fermentas (Hanover, Md.) high range RNA ladder; 1-6,
corresponding ripening stage of fruit.
[0061] FIG. 35. ELISA analysis of HN concentration in ripening CHN
tomato fruit. (a) Tomato line CHN-1. (b) Tomato line CHN-10. (c)
Tomato line CHN-12. (d) Tomato line CHN-32. Except in line CHN-12
where only stage 1 fruit had three reps, bars represent the mean of
3 samples from 3 different fruit, error bars indicate the standard
error of the mean.
[0062] FIG. 36. Western analysis of crude protein extracts from
wild type and transgenic tomato fruit and leaves and NT1 cell
extracts. NF, represents tomato fruit negative control--wild type
fruit; NL tomato leaf negative control--wild type leaf; NNT NT1
cell negative control--non-transformed cell lines; 119, transgenic
NT1 cell line 119; L10, leaf from transgenic tomato line 10; L32,
leaf from tomato line 32; HN, animal derived Lasota NDV virus; M,
Bio-Rad's precision plus protein all blue standard; 1-1, fruit from
line CHN-1, stage 1 of ripening; 1-3, fruit from line CHN-1, stage
3 of ripening; 1-6, fruit from line CHN-1, stage 6 of ripening;
32-1, fruit from line CHN-32, stage 1 of ripening; 32-3, fruit from
line CHN-32, stage 3 of ripening; 32-6, fruit from line CHN-32,
stage 6 of ripening; 10-1, fruit from CHN-10, stage 1 of ripening.
Protein size is give in kDa.
[0063] FIG. 37. Haemagglutination activity in the fruit and leaves
of CHN tomatoes.
[0064] FIG. 38. Change in maturing fruit diameter. "Week" indicates
the amount of time post pollination. Points indicate the mean of
three measurements while the bars indicate the standard errors of
the means.
[0065] FIG. 39. Change in fruit mass of maturing tomato fruit.
"Week" indicates the amount of time post pollination. Each point
represents the mean of the three measurements while the bars
indicate the standard errors of the means.
[0066] FIG. 40. Water loss from maturing tomato fruit upon
lyophilization. "Week" indicates the amount of time post
pollination. Points represent the mean of three measurements while
the bars indicate the standard errors of the means.
[0067] FIG. 41. Concentration of HN per gram of fresh tomato fruit.
"Week" indicates the amount of time post pollination. Bars
represent the average of three samples. Bars labeled with the same
letter are not significantly different (.alpha.=0.05). Error bars
indicate the standard error of the means.
[0068] FIG. 42. Amount of HN in maturing tomato fruit. "Week"
indicates the amount of time post pollination. Bars represent the
average of three replicate HN contents multiplied by the masses.
Bars labeled with the same letter are not significantly different
(.alpha.=0.05). Error bars indicate the standard error of the
mean.
[0069] FIG. 43. The regulated biological agent (PCHN) insert in
CHN-18 master seed.
[0070] FIG. 44. DNA sequence of the whole gene insert in CHN-18
master seed (SEQ ID NO: 12).
[0071] FIG. 45. pCHA vector sequence (SEQ ID NO: 24).
[0072] FIG. 46. pMHN vector sequence (SEQ ID NO: 25).
[0073] FIG. 47. pCHN vector sequence (SEQ ID NO: 26).
[0074] FIG. 48. Construction of pUHN.
SUMMARY OF THE SEQUENCES
[0075] SEQ ID NOS: 1 and 2, shown in FIG. 1, are the plant
optimized coding sequence and protein sequence of the HN gene of
NDV strain "Lasota".
[0076] SEQ ID NOS: 3 and 4, shown in FIG. 10, are the DNA and
protein sequences of the HA gene of AIV A/turkey/Wisconsin/68
(H5N9).
[0077] SEQ ID NO: 5 is a PCR primer used to end-tailor the CsVMV
promoter on pCP!H.
[0078] SEQ ID NO: 6 is a PCR primer used to end-tailor the CsVMV
promoter on pCP!H.
[0079] SEQ ID NO: 7 is a mutagenic primer used to create a Nco I
site.
[0080] SEQ ID NO: 8 is a forward primer complimentary to the 5'
region.
[0081] SEQ ID NO: 9 is a mutagenic primer used to create a XhoI I
site.
[0082] SEQ ID NO: 10 is a PCR labeled probe made by using the
primer HNa.
[0083] SEQ ID NO: 11 is a PCR labeled probe made by using the
primer HNb.
[0084] SEQ ID NO: 12 is the DNA sequence of the whole gene insert
in CHN-18 master seed.
[0085] SEQ ID NO: 13 is the DNA sequence encoding Hepatitis B virus
Strain Gly D surface antigen, complete cds. (GenBank accession
AF134148).
[0086] SEQ ID NO: 14 is the protein sequence of Hepatitis B virus
Strain Gly D surface antigen. (GenBank accession AAD31865).
[0087] SEQ ID NO: 15 is the DNA sequence encoding Homo sapiens zona
pellucida glycoprotein 3 (sperm receptor) (ZP3), mRNA. (GenBank
accession NM.sub.--007155).
[0088] SEQ ID NO: 16 is the protein sequence of Homo sapiens zona
pellucida glycoprotein 3 preproprotein (sperm receptor) (ZP3).
(GenBank accession NP.sub.--009086).
[0089] SEQ ID NO: 17 is the DNA sequence encoding Avian influenza
virus hemagglutinin (HA) mRNA, complete cds. (GenBank accession
U67783).
[0090] SEQ ID NO: 18 is the protein sequence of Avian influenza
virus hemagglutinin (HA). (GenBank accession AAC58999).
[0091] SEQ ID NO: 19 is the DNA sequence encoding Newcastle disease
virus hemagglutinin-neuraminidase (HN), mRNA, complete cds.
(GenBank accession AY510092).
[0092] SEQ ID NO: 20 is the protein sequence of Newcastle disease
virus hemagglutinin-neuraminidase (HN). (GenBank accession
AAS10195).
[0093] SEQ ID NO: 21 is the DNA sequence encoding Gallus gallus
zona pellucida glycoprotein 3 (sperm receptor) (ZP3), mRNA.
(GenBank accession NM.sub.--204389).
[0094] SEQ ID NO: 22 is the protein sequence of Gallus gallus zona
pellucida glycoprotein 3 (sperm receptor) (ZP3). (GenBank accession
NP.sub.--989720).
[0095] SEQ ID NO: 23 is the DNA sequence of Duck hepatitis B virus.
(GenBank accession X58569).
[0096] SEQ ID NO: 24 is the DNA sequence of vector pCHA.
[0097] SEQ ID NO: 25 is the DNA sequence of vector pMHN.
[0098] SEQ ID NO: 26 is the DNA sequence of vector pCHN.
DEFINITIONS
[0099] As used herein, "an immunogen or immunoprotective antigen"
is a non-self substance that elicits a humoral and/or cellular
immune response in healthy animals such that the animal is
protected against future exposure to a pathogen bearing the
immunogen. The pathogens are typically agents such as viruses,
bacteria, fungi and protozoa. Immunogens may also be antigenic
portions of pathogens including cell wall components and viral coat
proteins.
[0100] As used herein, "an immunoprotective particle" is a particle
or vesicle derived from a transgenic plant cell that expresses an
immunogen that, when appropriately administered to an animal,
provides protection against future exposure to a pathogen bearing
the immunogen.
[0101] As used herein, "vaccination or vaccinating" is defined as a
means for providing protection against a pathogen by inoculating a
host with an immunogenic preparation of a pathogenic agent, or a
non-virulent form or part thereof, such that the host immune system
is stimulated and prevents or attenuates subsequent host reactions
to later exposures of the pathogen. "Providing protection" refers
to stimulating an immune response as defined hereinbelow.
[0102] As used herein, "a vaccine" is a composition used to
vaccinate an animal that contains at least one immunoprotective
antigenic substances.
[0103] As used herein, "a pathogenic organism" is a bacterium,
virus, fungus, or protozoan that causes a disease or medical
condition in an animal which it has infected.
[0104] As used herein, "an adjuvant" is a substance that
accentuates, increases, or enhances the immune response to an
immunogen or antigen. As used herein, an increase, or accentuation
or enhancement means a 2-fold or more, for example, 2, 3, 4, 5, 10,
20, 30, 40, 50, 60, 70, 80, 90, 100, or 1000-fold or more increase
in the amount of antibody produced, for example, in the response to
an antigen administered in the presence of an adjuvant as compared
to in the absence of an adjuvant. An increase, accentuation or
enhancement also means at least 5% or more antibody production, for
example, 5, 6, 10, 20, 30, 40, 50, 60 70, 80, 90 or 100% or more,
for example, in response to an antigen administered in the presence
versus the absence of an adjuvant. Adjuvants typically enhance both
the humoral and cellular immune response but an increased response
to either in the absence of the other qualifies to define an
adjuvant. Moreover, adjuvants and their uses are well known to
immunologists and are typically employed to enhance the immune
response when doses of immunogen are limited or when the immunogen
is poorly immunogenic or when the route of administration is
sub-optimal. Thus the term `adjuvanting amount` is that quantity of
adjuvant capable of enhancing the immune response to a given
immunogen or antigen. The mass that equals an adjuvanting amount
will vary and is dependant on a variety of factors including but
not limited to the characteristics of the immunogen, the quantity
of immunogen administered, the host species, the route of
administration, and the protocol for administering the immunogen.
The adjuvanting amount can readily be quantified by routine
experimentation given a particular set of circumstances. This is
well within the ordinarily skilled artisan's purview and typically
employs the use of routine dose response determinations to varying
amounts of administered immunogen and adjuvant. Responses are
measured by determining serum antibody titers raised in response to
the immunogen using enzyme linked immunosorbant assays, radio
immune assays, hemagglutination assays and the like.
[0105] As used herein, a "transgenic plant cell" refers to a plant
cell which stably expresses a foreign gene, wherein the foreign
gene is integrated into the plant cell chromosome and does not
carry with it a viral vector sequence unique to a virus, where the
foreign gene is passed onto the next cell generation and is capable
of being expressed from the host plant cell chromosome. In
addition, "transgenic plant material" refers to a "transgenic cell
suspension" comprising one or a plurality of "transgenic plant
cells" obtained by well-known cell culture techniques (Street, HE.
1973, Plant tissue and cell culture: botanical monographs. Vol II,
University of California, Berkeley).
[0106] As used herein, a "trangenic plant" refers to a plant, the
cells of which stably express a "heterologous" foreign gene,
wherein the foreign gene is integrated into the plant cell
chromosome and does not carry with it a viral vector sequence
unique to a virus, where the foreign gene is passed onto the next
plant generation and is capable of being expressed from the host
plant cell chromosome. A "transgenic plant" comprises a "plurality
of transgenic plant cells". A "transgenic plant" refers to the
whole plant, or a part thereof including, but not limited to roots,
stems, leaves, stalks, seeds, fruit, tubers, flowers, pollen, and
the like. Examples of heterologous foreign genes include, but are
not limited to, Norwalk virus capsid protein (NVCP), Avian
Influenza hemagglutination antigen (AIV-HA), Newcastle Disease
Virus neuraminidase (NDV-HN), zona pellucida glycoprotein 3 (ZP3),
and Hepatitis B surface Antigen (HBsAg).
[0107] Transgenic plant is herein defined as a plant cell culture,
plant cell line, plant, or progeny thereof derived from a
transformed plant cell or protoplast, wherein the genome of the
transformed plant contains foreign DNA, introduced by laboratory
techniques, not originally present in a native, non-transgenic
plant cell of the same species. The terms "transgenic plant" and
"transformed plant" have sometimes been used in the art as
synonymous terms to define a plant whose DNA contains an exogenous
DNA molecule.
[0108] As used herein, an "edible plant" refers to a plant which
may be consumed by an animal, has nutritional value and is not
toxic. An "edible plant" may be a "food" which is a plant or a
material obtained from a plant which is ingested by humans or other
animals. The term "food" is intended to include plant material
which may be fed to humans and other animals or a processed plant
material which is fed to humans and other animals. Materials
obtained from a plant are intended to include a component of a
plant which is eventually ingested by a human or other animal.
Examples of "edible plant" include, but are not limited to, tomato
plants, rice plants, wheat plants, corn plants, carrot plants,
potato plants, apple plants, soybean plants, alfalfa plants,
medicago plants, vegetable plants, and fruit plants or any of the
edible plants described herein.
[0109] In some cases an "edible plant" is "capable of being
ingested for its nutritional value", which refers to a plant or
portion thereof that provides a source of metabolizable energy,
supplementary or necessary vitamins or co-factors, roughage or
otherwise beneficial effect upon ingestion by an animal. Thus,
where the animal to be treated by the methods of the present
invention is an herbivore capable of bacterial-aided digestion of
cellulose, such a food might be represented by a transgenic grass
plant. Other edible plants include vegetables and fruits.
Similarly, although transgenic lettuce plants, for example, do not
substantially contribute energy sources, building block molecules
such as proteins, carbohydrates or fats, nor other necessary or
supplemental vitamins or cofactors, a lettuce plant transgenic for
the nucleic acid molecules described herein used as food for an
animal would fall under the definition of a food as used herein if
the ingestion of the lettuce contributed roughage to the benefit of
the animal, even if the animal could not digest the cellulosic
content of lettuce. An "edible plant" therefore excludes
tobacco.
[0110] As used herein, "immune response" refers to a response made
by the immune system of an organism to a substance, which includes
but is not limited to foreign or self proteins. There are three
general types of "immune response" including, but not limited to
mucosal, humoral and cellular "immune responses." A "mucosal immune
response" results from the production of secretory IgA (sIga)
antibodies in secretions that bathe all mucosal surfaces of the
respiratory tract, gastrointestinal tract and the genitourinary
tract and in secretions from all secretory glands (McGhee, J. R. et
al., 1983, Annals NY Acad. Sci. 409). These sIgA antibodies act to
prevent colonization of pathogens on a mucosal surface (Williams,
R. C. et al., Science 177, 697 (1972); McNabb, P. C. et al., Ann.
Rev. Microbiol. 35, 477 (1981)) and thus act as a first line of
defense to prevent colonization or invasion through a mucosal
surface. The production of sIgA can be stimulated either by local
immunization of the secretory gland or tissue or by presentation of
an antigen to either the gut-associated lymphoid tissue (GALT or
Peyer's patches) or the bronchial-associated lymphoid tissue (BALT;
Cebra, J. J. et al., Cold Spring Harbor Symp. Quant. Biol. 41, 210
(1976); Bienenstock, J. M., Adv. Exp. Med. Biol. 107, 53 (1978);
Weisz-Carrington, P. et al., J. Immunol 123, 1705 (1979);
McCaughan, G. et al., Internal Rev. Physiol 28, 131 (1983)).
Membranous microfold cells, otherwise known as M cells, cover the
surface of the GALT and BALT and may be associated with other
secretory mucosal surfaces. M cells act to sample antigens from the
luminal space adjacent to the mucosal surface and transfer such
antigens to antigen-presenting cells (dendritic cells and
macrophages), which in turn present the antigen to a T lymphocyte
(in the case of T-dependent antigens), which process the antigen
for presentation to a committed B cell. B cells are then stimulated
to proliferate, migrate and ultimately be transformed into an
antibody-secreting plasma cell producing IgA against the presented
antigen. When the antigen is taken up by M cells overlying the GALT
and BALT, a generalized mucosal immunity results with sIgA against
the antigen being produced by all secretory tissues in the body
(Cebra et al., supra; Bienenstock et al., supra; Weinz-Carrington
et al., supra; McCaughan et al., supra). Oral immunization is
therefore an important route to stimulate a generalized mucosal
immune response and, in addition, leads to local stimulation of a
secretory immune response in the oral cavity and in the
gastrointestinal tract.
[0111] An "immune response" may be measured using techniques known
to those of skill in the art. For example, serum, blood or other
secretions may be obtained from an organism for which an "immune
response" is suspected to be present, and assayed for the presence
of the above mentioned immunoglobulins using an enzyme-linked
immuno-absorbant assay (ELISA; U.S. Pat. No. 5,951,988; Ausubel et
al., Short Protocols in Molecular Biology 3d Ed. John Wiley &
Sons, Inc. 1995). According to the present invention, a protein of
the present invention can be said to stimulate an "immune response"
if the quantitative measure of immunoglobulins in an animal treated
with a protein of interest detected by ELISA is statistically
different (for example, is increased or decreased by 2-fold or
more, for example, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90,
100, or 1000-fold or more increase or decrease in the amount of
antibody produced. An increase or decrease also means at least 5%
or more antibody production, for example, 5, 6, 10, 20, 30, 40, 50,
60 70, 80, 90 or 100% or more, or at least 5% or more of a decrease
in antibody production) from the measure of immunoglobulins
detected in an animal not treated with a protein of interest,
wherein said immunoglobulins are specific for the protein of
interest. A statistical test known in the art and useful to
determining the difference in measured immunoglobulin levels
includes, but is not limited to ANOVA, Student's T-test, and the
like, wherein the P value is at least <0.1, <0.05, <0.01,
<0.005, <0.001, and even <0.0001.
[0112] An "immune response" may be measured using other techniques
such as immunohistochemistry using labeled antibodies which are
specific for portions of the immunoglobulins raised during the
"immune response". Tissue (e.g., ovarian tissue) from an animal to
which a protein of interest has been administered according to the
invention may be obtained and processed for immunohistochemistry
using techniques well known in the art (Ausubel et al., Short
Protocols in Molecular Biology 3d Ed. John Wiley & Sons, Inc.
1995). Microscopic data obtained by immunohistochemistry may be
quantitated by scanning the immunohistochemically stained tissue
sample and quantitating the level of staining using a computer
software program known to those of skill in the art including, but
not limited to NIH Image (National Institutes of Health, Bethesda,
Md.). According to the present invention, a protein of the present
invention can be said to stimulate an "immune response" if the
quantitative measure of immunohistochemical staining in an animal
treated with a protein of interest is statistically different (as
defined by an increase or decrease discussed hereinabove) from the
measure of immunohistochemical staining detected in an animal not
treated with the protein of interest, wherein said histochemical
staining requires binding specific for that protein. A statistical
test known in the art may be used to determine the difference in
measured immunohistochemical staining levels including, but not
limited to ANOVA, Student's T-test, and the like, wherein the P
value is at least <0.1, <0.05, <0.01, <0.005,
<0.001, and even <0.0001.
[0113] A "mucosal immune response" may be "detected" using any of
the above referenced techniques. For example, an ELISA assay may be
employed using anti-IgA antibodies to detect and measure the
mucosal-specific immunoglobulins (Dickinson, B. L. & Clements,
J. D. Dissociation of Escherichia coli heat-labile enterotoxin
adjuvanticity from ADP-ribosyltransferase activity. Infect Immun
63, 1617-1623 (1995)).
[0114] A "humoral immune response" comprises the production of
antibodies in response to an antigen or antigens. A cellular immune
response includes responses such as a helper T-cell (CD4.sup.+)
response and a cytotoxic T-cell lymphocyte (CD8.sup.+) response. A
mucosal immune response (or secretory immune response) comprises
the production of secretory (sIga) antibodies. An immune response
can comprise one or a combination of these responses.
[0115] As used herein, "animal" refers to an organism classified
within the phylogenetic kingdom Animalia. As used herein, an
"animal" also refers to a mammal. Animals, useful in the present
invention, include, but are not limited to mammals, marsupials,
mice, dogs, cats, cows, humans, deer, horses, sheep, livestock,
poultry, chickens, turkeys, ostrich, fish, fin fish, shell fish,
and the like.
[0116] As used herein, "monocotyledonous" refers to a type of plant
whose embryos have one cotyledon or seed leaf. Examples of
"monocots" include, but are not limited to lilies; grasses; corn;
grains, including oats, wheat and barley; orchids; irises; onions
and palms.
[0117] As used herein, "dicotyledonous" refers to a type of plant
whose embryos have two seed halves or cotyledons. Examples of
"dicots" include, but are not limited to tobacco; tomato; the
legumes including alfalfa; oaks; maples; roses; mints; squashes;
daisies; walnuts; cacti; violets and buttercups.
[0118] As used herein, "vector" refers to a nucleic acid molecule
capable of transporting another nucleic acid to which it has been
linked. One type of vector is a "plasmid", which refers to a
circular double stranded nucleic acid loop into which additional
nucleic acid segments can be ligated. Certain vectors are capable
of autonomous replication in a host cell into which they are
introduced (e.g., bacterial vectors having a bacterial origin of
replication and episomal mammalian vectors). Other vectors (e.g.,
non-episomal mammalian vectors) are integrated into the genome of a
host cell upon introduction into the host cell, and thereby are
replicated along with the host genome. Moreover, certain vectors
are capable of directing the expression of genes to which they are
operatively linked. Such vectors are referred to herein as
"expression vectors". In general, expression vectors of utility in
recombinant nucleic acid techniques are often in the form of
plasmids. In the present specification, "plasmid" and "vector" can
be used interchangeably as the plasmid is the most commonly used
form of vector.
[0119] As used herein, "promoter" refers to a sequence of DNA,
usually upstream (5') of the coding region of a structural gene,
which controls the expression of the coding region by providing
recognition and binding sites for RNA polymerase and other factors
which may be required for initiation of transcription. The
selection of the promoter will depend upon the nucleic acid
sequence of interest. A "plant-functional promoter" refers to a
"promoter" which is capable of supporting the initiation of
transcription in plant cells. "Plant-functional promoters" useful
in the present invention include, but are not limited to the 35S
promoter of the cauliflower mosaic virus (CaMV); promoters of seed
storage protein genes such as Zma10Kz or Zmag12, light inducible
genes such as ribulose bisphosphate carboxylase small subunit
(rbcS), stress induced genes such as alcohol dehydrogenase (Adh1),
or "housekeeping genes" that express in all cells (such as Zmact, a
maize actin gene); the tomato E8 promoter; ubiquitin; mannopine
synthetase (mas); rice actin 1; soybean seed protein glycinin
(Gy1); soybean vegetative storage protein (vsp); and granule-bound
starch synthase (gbss). Other "plant-functional promoters" include
promoters for genes which are known to give high expression in
edible plant parts, such as the patatin gene promoter from
potato.
[0120] As used herein, "operably linked" refers to a juxtaposition
wherein the components described are in a relationship permitting
them to function in their intended manner. A control sequence
"operably linked" to a coding sequence is ligated in such a way
that expression of the coding sequence is achieved under conditions
compatible with the control sequences. A promoter sequence is
"operably-linked" to a gene when it is in sufficient proximity to
the transcription start site of a gene to regulate transcription of
the gene.
[0121] As used herein, "administered" refers to the delivery of the
transgenic plant material, cells, compositions, and pharmaceutical
formulations of the present invention to an animal in such a manner
so to guarantee that the "delivered" material contacts a mucosal
surface of the animal to which it was administered. Routes of
"delivery" useful in the present invention include, but are not
limited to oral delivery, nasal delivery, intraperitoneal delivery,
intramuscular, intravenous or subcutaneous delivery rectal or
vaginal delivery (e.g., by suppository, or topical administration),
or a route of delivery wherein the delivered material directly
contacts a mucosal surface (i.e., "mucosal delivery"). As used
herein, "pharmaceutically acceptable" means a non-toxic material
that does not interfere with the effectiveness of the biological
activity of the active ingredient(s). The characteristics of the
carrier will depend on the route of administration.
[0122] As used herein, a "mucosal surface", "mucosal membrane", or
"mucosa" refers to the well known medical definition of these
structures, which is the surface or lining of a structure
comprising an epithelium, lamina propria, and, in the digestive
tract, a layer of smooth muscle. Examples of "mucosal surfaces"
include, but are not limited to the inner coat of the bronchi, the
mucous layer of the tympanic cavity, the inner mucous coat of the
colon, the inner layer of the ductus deferens, the inner coat of
the esophagus, the mucous coat of the small intestine, the mucous
coat of the larynx, the mucous membrane of the tongue, the
pituitary membrane, the mucous membrane of the oral cavity, the
mucous membrane of the pharynx, the inner mucous layer of the
trachea, the lining of the auditory tube, the mucous layer of the
uterine tube, the inner layer of the ureter, the inner layer of the
urethra, the endometrium, the mucous membrane of the vagina, the
mucous layer of the stomach, the inner coat of the urinary bladder,
and the mucous membrane of the seminal vesicle.
[0123] As used herein, a "carrier" refers to an inert and non-toxic
material suitable for accomplishing or enhancing delivery of the
vaccine of the present invention into an animal. Examples of a
carrier include, but are not limited to water, phosphate buffered
saline, or saline, and further may include an adjuvant. Adjuvants
such as incomplete Freund's adjuvant, aluminum phosphate, aluminum
hydroxide, or alum are materials well known in the art.
[0124] The present invention also provides pharmaceutical and
veterinary compositions comprising an immunoprotective particle of
the present invention in combination with one or more
pharmaceutically acceptable adjuvants carriers, diluents, and
excipients. Such pharmaceutical compositions may also be referred
to as vaccines and are formulated in a manner well known in the
pharmaceutical vaccine arts.
[0125] "Administering" or "administer" is defined as the
introduction of a substance into the body of an animal and includes
oral, nasal, rectal, vaginal and parenteral routes. The claimed
compositions may be administered individually or in combination
with other therapeutic agents via any route of administration,
including but not limited to subcutaneous (SQ) intramuscular (IM),
intravenous (IV), mucosal, nasal or oral. The compositions may be
administered via the SQ or IM route. Especially preferred is the
mucosal route, and most preferred is the oral route.
[0126] As used herein, "an effective amount or dosage of the
vaccine" is an amount necessary to stimulate an innate immune
response as defined herein and as detected by the assays described
herein as in a human or animal sufficient for the human or animal
to effectively resist a challenge mounted by a pathogen. For
example, in one embodiment, "an effective amount or dosage of the
vaccine" causes an increase in the amount of antibody that binds to
the immunoprotective antigen of the vaccine. As used herein, an
increase means a 2-fold or more, for example, 2, 3, 4, 5, 10, 20,
30, 40, 50, 60, 70, 80, 90, 100, or 1000-fold or more increase in
the amount of antibody produced by the vaccinated subject as
compared to an unvaccinated subject. An increase also means at
least 5% or more antibody production, for example, 5, 6, 10, 20,
30, 40, 50, 60 70, 80, 90 or 100% or more, by a vaccinated subject
as compared to an unvaccinated subject. The dosages administered to
such human or animal will be determined by a physician or
veterinarian in light of the relevant circumstances including the
particular immunoprotective particle or combination of particles,
the condition of the human or animal, and the chosen route of
administration. The dosage ranges presented herein are not intended
to limit the scope of the invention in any way and are presented as
general guidance for the skilled practitioner. The effective dosage
can be estimated initially either in cell culture assays, or in
animal models, usually mice, rabbits, dogs, or pigs. The animal
model is also used to achieve a desirable concentration range and
route of administration. Such information can then be used to
determine useful dosages and routes for administration in
humans.
[0127] The exact dosage is chosen by the individual physician in
view of the patient to be treated. Dosage and administration are
adjusted to provide sufficient levels of the active moiety or to
maintain the desired effect. Additional factors which may be taken
into account include the severity of the disease state; age, weight
and gender of the subject; diet, time and frequency of
administration, drug combination(s), reaction sensitivities, and
tolerance/response to therapy. Long acting pharmaceutical
compositions might be administered every 3 to 4 days, every week,
or once every two weeks depending on half-life and clearance rate
of the particular formulation.
[0128] The particular dosages of an antigenic composition of the
invention will depend on many factors including, but not limited to
the species, age, and general condition of the human or animal to
which the composition is administered, and the mode of
administration of the composition. An effective amount of the
composition of the invention can be readily determined using only
routine experimentation. In vitro and in vivo models (for example
poultry) can be employed to identify appropriate doses. Generally,
0.1, 1.0, 1.5, 2.0, 5, 10, or 100 mg/kg of an antigen will be
administered to a large mammal, such as a baboon, chimpanzee, or
human. If desired, co-stimulatory molecules or adjuvants can also
be provided before, after, or together with the antigenic
compositions. Preferably, the dosage of antigen is administered in
the range of 1 ng to 0.5 mg/kg bodyweight, more preferably, 1 mg to
50 mg/kg of body weight.
[0129] The efficacy of an edible vaccine according to the invention
is determined by demonstrating that the administration of the
vaccine prevents or ameliorates the symptoms of the disease being
treated or caused by the pathogen of interest, by at least 5%,
preferably 10-20% and more preferably, 25-100%.
[0130] "Bird" is herein defined as any warm-blooded vertebrate
member of the class Aves having forelimbs modified into wings,
scaly legs, a beak, and bearing young in hard-shelled eggs. For
purposes of this specification, preferred groups of birds are
domesticated chickens, turkeys, ostriches, ducks, geese, and
cornish game hens. A more preferred group is domesticated chickens
and turkeys. The most preferred bird for purposes of this invention
is the domesticated chicken, including broilers and layers.
[0131] The methods and compositions of the present invention are
directed toward immunizing and protecting humans and animals,
preferably domestic animals, such as birds (poultry), cows, sheep,
goats, pigs, horses, cats, dogs and llamas, and most preferably
birds. Certain of these animal species can have multiple stomachs
and digestive enzymes specific for the decomposition of plant
matter, and may otherwise readily inactivate other types of oral
vaccines. While not meant to be a limitation of the invention,
ingestion of transgenic plant cells, and compositions derived
therefrom, can result in immunization of the animals at the site of
the oral mucosa including the tonsils.
[0132] As used herein, "fruit" refers to the ovary of an angiosperm
flower and the associated structures (e.g. the receptacle or parts
of the floral tube) that enlarge and develop to form a mass of
tissue surrounding the seeds. According to the invention, the
particular tissues that are involved in fruit development vary with
the species, but tissues involved in fruit development according to
the invention, are always derived from the maternal parent of the
progeny seeds.
[0133] As used herein, "ripe" refers to a stage of fruit
development that is characterized by changes in pigmentation, the
conversion of acids and starches to free sugars, and breakdown of
cell walls that results in softening of the fruit.
[0134] As used herein, "fruit ripening conditions" refer to
conditions under which the developmental processes involved in
fruit ripening can occur, including cell division and expansion of
maternal tissues that occurs after fertilization of ovaries. As
used herein, for example, production of ethylene is a chemical
signal that stimulates the genetic program for ripening in
climacteric fruits such as tomato.
[0135] As used herein, "prior to the onset of fruit ripening"
refers to a stage in fruit development wherein less than 10% (for
example, 9.9, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5%) of the fruit has
undergone a change in pigmentation. "Prior to the onset of fruit
ripening" also refers to a stage in fruit development wherein less
than 10% (for example, 9.9, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5%) of the
acids and starches of a fruit are converted to sugar. "Prior to the
onset of fruit ripening" also refers to a stage in fruit
development wherein less than 10% (for example, 9.9, 9, 8, 7, 6, 5,
4, 3, 2, 1, 0.5%) of the cell wall material of a fruit is
degraded.
[0136] As used herein, "incubating" includes growing a plant either
in the field or in a controlled or uncontrolled laboratory or
indoor setting. In one embodiment of the invention, an antigen is
produced in a plant by "incubating", as defined herein, the plant
under conditions wherein said plant expresses the antigen prior to
the onset of fruit ripening.
DETAILED DESCRIPTION OF THE INVENTION
[0137] The invention relates to sequences encoding an antigen of
interest, for example a plant optimized sequence encoding HN
antigen of Newcastle Disease Virus or HA antigen of Avian Influenza
Virus. The invention also relates to vectors, plant cells,
transgenic plants and vaccines comprising the plant optimized
sequences of the invention. The invention further relates to
methods of protecting against viral infection, for example
infection by Newcastle Disease Virus of Avian Influenza Virus. The
invention also relates to methods of antigen production in
transgenic plants.
Immunoprotective Antigens Useful According to the Invention
[0138] The invention provides for plant cells and transgenic plants
expressing a heterologous foreign gene. A heterologous foreign gene
of the invention can be any gene of interest including but not
limited to Norwalk virus capsid protein (NVCP) (Genbank Accession
Number: M87661, GenBank #AF093797, Genome for Norwalk Virus,
Genbank Accession Number AAB50466, for NV capsid protein), Avian
Influenza hemagluttination antigen (AIV-HA) (Genbank Accession
Number U67783 and AAC58999), Newcastle Disease Virus neuraminidase
(NDV-HN) (Genbank Accession Numbers NM-204389, NP-989720,
NP-009086, and NM-007155) (Genbank Accession Number: AY510092 and
AAS10195), zona pellucida glycoprotein 3(ZP3), Hepatitis B surface
Antigen (HBsAg) (Genbank Accession Numbers AF134148, AAD31865,
X58569, GenBank #AF090842), shigatoxin B (StxB) (Genbank
#AJ132761), staphylococcus enterotoxin B (SEB)(GenBank #M11118), E.
coli labile toxin B (LT-B)(GenBank#AB01677), and E. coli labile
toxin A subunit (LT-A) (GenBank #AB011677).
[0139] Newcastle's disease virus (NDV) is a member of the
Paramyxovirus genus of the Paramyxoviridae. Viruses in this genus
are enveloped negative-strand RNA viruses that also include
parainfluenza viruses like Sendai, respiratory syncytial, mumps and
measles viruses (Kingsbury et al., 1978, Intervirology,
10:137-152). Virions are characterized by the presence of two
surface glycoproteins including hemagglutinin neuraminidase (HN) a
74 kDA protein and a smaller fusion (F) protein. HN is involved in
two important functions including cell attachment by recognition of
sialic acid containing cell receptors, and neuraminidase activity
cleaving sialic acid from progeny virus particles to prevent
self-agglutination. The F protein mediates virus-to-cell and
cell-to-cell fusion and hemolysis. See Scheid, A., and Choppin, P.
W. (1973) J. Virology. 11, 263-271; Scheid, A, and Choppin, P. W.
(1974) Virology 57, 470-490; Lamb, R. A., and Kolakofsky, D.
(1996). Paramyxoviridae: the viruses and their replication, p.
577-604. In B. N. Fields, D. M. Knipe, and P. M. Howley (ed.),
Fields virology, 3.sup.rd ed. Lippincott-Raven Publishers,
Philadelphia, Pa. Polyvalent sera prepared against either protein
are capable of neutralizing the infectivity of the virus. See
Mertz, D. C., Scheid, A., and Choppin, P. W. (1980) J. Exp. Med.
151, 275-288.
[0140] Avian influenza virus is described in Suarez et al., Virus
Res. 1997, 51:115 and Sockett, Can. Med. Assoc. J., 1998, 158:369,
incorporated herein by reference in their entirety. The
hemagglutinin gene of avian influenza virus is described in Barun
et al., 1998, Nuc. Acids. Res., 16:4181, incorporated herein by
reference in its entirety.
Preparation of the Constructs of the Invention
[0141] An expression cassette according to the invention comprises
a DNA sequence encoding at least one immunoprotective antigen
operably linked to transcriptional and translational control
regions functional in a plant cell. Preferably the invention
provides plant expression cassettes that are useful for expressing
immunoprotective antigen transgenes in plants. These cassettes
comprise the following elements that are operably linked from 5' to
3':
[0142] A) a plant gene promoter sequence that naturally expresses
in plants;
[0143] B) a nucleic acid sequence encoding an immunoprotective
antigen of interest; and
[0144] C) a 3'UTR.
[0145] Promoters useful in this embodiment are any known promoters
that are functional in a plant. Many such promoters are well known
to the ordinarily skilled artisan. Such promoters include promoters
normally associated with other genes, and/or promoters isolated
from any bacterial, viral, eukaryotic, or plant cell. It may be
advantageous to employ a promoter that effectively directs the
expression of the foreign coding sequence in the cell or tissue
type chosen for expression. The use of promoter and cell type
combinations for protein expression is generally known to those of
skill in the art of molecular biology, for example, see Sambrook et
al., In: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y., 1989. The promoters employed
may be constitutive, or inducible, and can be used 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 or peptides. The term
"constitutive" used in the context of a promoter means that the
promoter is capable of directing transcription of an operably
linked nucleic acid sequence in the absence of a stimulus (e.g.,
heat shock, chemicals, etc.). In contrast, an "inducible" promoter
is one which is capable of directing a level of transcription of an
operably linked nucleic acid sequence in the presence of a stimulus
(e.g., heat shock, chemicals, etc.), wherein the level of the
transcription is different from that in the absence of the
stimulus. As used herein, "inducible" also refers to expressed in
the presence of an exogenous or endogenous chemical (for example an
alcohol, a hormone, or a growth factor), in the presence of light
and/or in response to developmental changes. As used herein,
"inducible" also refers to expressed in any tissue in the presence
of a chemical inducer". As used herein, "chemical induction"
according to the invention refers to the physical application of a
exogenous or endogenous substance (including macromolecules e.g.
proteins, or nucleic acids) to a plant or a plant organ (e.g. by
spraying a liquid solution comprising a chemical inducer on leaves,
application of a liquid solution to roots or exposing plants or
plant organs to gas or vapor) which has the effect of causing the
target promoter present in the cells of the plant or plant organ to
increase the rate of transcription.
[0146] Some exemplary plant functional promoters, which can be used
to express a structural gene of the present invention, are among
the following: CaMV 35S and 19S promoters (U.S. Pat. No. 5,352,605
and U.S. Pat. No. 5,530,196); patatin promoter (U.S. Pat. No.
5,436,393); a B33 promoter sequence of a patatin gene derived from
Solanum tuberosum, and which leads to a tuber specific expression
of sequences fused to the B33 promoter (U.S. Pat. No. 5,436,393);
tomato E8 promoter (WO 94/24298); tomato fruit promoters (U.S. Pat.
No. 5,556,653); -a plant ubiquitin promoter system (U.S. Pat. Nos.
5,614,399 and 5,510,474); 5 cis-regulatory elements of abscisic
acid-responsive gene expression (U.S. Pat. No. 5,824,865); promoter
from a badnavirus, rice tungro bacilliform virus (RTBV) (U.S. Pat.
No. 5,824,857); a chemically inducible promoter fragment from the
5' flanking region adjacent the coding-region of a tobacco PR-1a
gene (U.S. Pat. No. 5,789,214); a raspberry drul promoter (U.S.
Pat. No. 5,783,394); strawberry promoters and genes (WO 98/31812);
promoter is the napin promoter, the phaseolin promoter, and the DC3
promoter (U.S. Pat. No. 5,773,697); a LEA promoter (U.S. Pat. No.
5,723,765); 5' transcriptional regulatory region for sink organ
specific expression (U.S. Pat. No. 5,723,757); G-box related
sequence motifs, specifically Iwt and PA motifs, which function as
cis-elements of promoters, to regulate the expression of
heterologous genes in transgenic plants (U.S. Pat. No. 5,723,751);
P119 promoters and their use (U.S. Pat. No. 5,633,440); Group 2
(Gp2) plant promoter sequences (U.S. Pat. No. 5,608,144); nucleic
acid promoter fragments derived from several genes from corn,
petunia and tobacco (U.S. Pat. No. 5,608,143); promoter sequences
isolated from the nuclear gene for chloroplast GS2 glutamine
synthetase and from two nuclear genes for cytosolic GS3 glutamine
synthetase in the pea plant, Pisum sativum (U.S. Pat. No.
5,391,725); full-length transcript promoter from figwort mosaic
virus (FMV) (U.S. Pat. No. 5,378,619); an isocitrate lyase promoter
(U.S. Pat. No. 5,689,040); a microspore-specific regulatory element
(U.S. Pat. No. 5,633,438); expression of heterologous genes in
transgenic plants and plant cells using plant asparagine synthetase
promoters (U.S. Pat. No. 5,595,896); a promoter region that drives
expression of a 1450 base TR transcript in octopine-type crown gall
tumors (U.S. Pat. No. 4,771,002); promoter sequences from the gene
from the small subunit of ribulose-1,5-bisphosphate carboxylase
(U.S. Pat. No. 4,962,028); the Arabidopsis histone H4 promoter
(U.S. Pat. No. 5,491,288); a seed-specific plant promoter (U.S.
Pat. No. 5,767,363); a 21 bp promoter element which is capable of
imparting root expression capability to a rbcS-3A promoter,
normally a green tissue specific promoter (U.S. Pat. No.
5,023,179); promoters of tissue-preferential transcription of
associated DNA sequences in plants, particularly in the roots (U.S.
Pat. No. 5,792,925); Brassica sp. polygalacturonase promoter (U.S.
Pat. No. 5,689,053); a seed coat-specific cryptic promoter region
(U.S. Pat. No. 5,824,863); a chemically inducible nucleic acid
promoter fragment isolated from the tobacco PR-1a gene inducible by
application of a benzo-1,2,3-thiadiazole, an isonicotinic acid
compound, or a salicylic acid compound (U.S. Pat. No. 5,689,044);
promoter fragment isolated from a cucumber chitinase/lysozyme gene
that is inducible by application of benzo-1,2,3-thiadiazole (U.S.
Pat. No. 5,654,414); a constitutive promoter from tobacco that
directs expression in at least ovary, flower, immature embryo,
mature embryo, seed, stem, leaf and root tissues (U.S. Pat. No.
5,824,872); alteration of gene expression in plants (U.S. Pat. No.
5,223,419); a recombinant promoter for gene expression in
monocotyledenous plants (U.S. Pat. No. 5,290,924); method for using
TMV to overproduce peptides and proteins (WO 95/21248); nucleic
acid comprising shoot meristem-specific promoter and regulated
sequence (WO 98/05199); phaseolin promoter and structural gene
(EP-B-0122791); plant promoters [sub domain of CaMV .sup.35S] (U.S.
Pat. No. 5,097,025); use of tomato E8-derived promoters to express
heterologous genes, e.g. 5-adenosylmethionine hydrolase in ripening
fruit (WO 94/24294); method of using transactivation proteins to
control gene expression in transgenic plants (U.S. Pat. No.
5,801,027); DNA molecules encoding inducible plant promoters and
tomato Adh2 enzyme (U.S. Pat. No. 5,821,398); synthetic plant core
promoter and upstream regulatory element (WO 97/47756); monocot
having dicot wound inducible promoter (U.S. Pat. No. 5,684,239);
selective gene expression in plants (U.S. Pat. No. 5,110,732); CaMV
35S enhanced mannopine synthase promoter and method for using the
same (U.S. Pat. No. 5,106,739); seed specific transcription
regulation (U.S. Pat. No. 5,420,034); seed specific promoter region
(U.S. Pat. No. 5,623,067); DNA promoter fragments from wheat (U.S.
Pat. No. 5,139,954); chimeric regulatory regions and gene cassettes
for use in plants (WO 95/14098); production of gene products to
high levels (WO 90/13658); HMG promoter expression system and post
harvest production of gene products in plants and plant cell
cultures (U.S. Pat. No. 5,670,349); gene expression system
comprising the promoter region of the alpha amylase genes in plants
(U.S. Pat. No. 5,712,112).
[0147] A preferred group of promoters is the cassaya vein mosaic
virus promoters described in U.S. patent application Ser. No.
09/202,838, herein incorporated by reference in its entirety; the
phaseolin promoters described in U.S. Pat. No. 5,591,605, herein
incorporated by reference in its entirety; rice actin promoters
described in U.S. Pat. No. 5,641,876, herein incorporated by
reference in its entirety; the per5 promoter described in WO
98/56921, herein incorporated by reference in its entirety; and the
gamma zein promoters described in WO 00/12681.
[0148] A promoter DNA sequence is said to be "operably linked" to a
coding DNA sequence if the two are situated such that the promoter
DNA sequence influences the transcription of the coding DNA
sequence. For example, if the coding DNA sequence codes for the
production of a protein, the promoter DNA sequence would be
operably linked to the coding DNA sequence if the promoter DNA
sequence affects the expression of the protein product from the
coding DNA sequence.
[0149] Construction of gene cassettes is readily accomplished
utilizing well known methods, such as those disclosed in Sambrook
et al. (1989); and Ausubel et al. (1987) Current Protocols in
Molecular Biology, John Wiley and Sons, New York, N.Y. The present
invention also includes DNA sequences having substantial sequence
homology with the disclosed sequences encoding immunoprotective
antigens such that they are able to have the disclosed effect on
expression. As used in the present application, the term
"substantial sequence homology" is used to indicate that a
nucleotide sequence (in the case of DNA or RNA) or an amino acid
sequence (in the case of a protein or polypeptide) exhibits
substantial, functional or structural equivalence with another
nucleotide or amino acid sequence. Any functional or structural
differences between sequences having substantial sequence homology
will be de minimis; that is they will not affect the ability of the
sequence to function as indicated in the present application.
Sequences that have substantial sequence homology with the
sequences disclosed herein are usually variants of the disclosed
sequence, such as mutations, but may also be synthetic
sequences.
[0150] In most cases, sequences having 95% homology to the
sequences specifically disclosed herein will function as
equivalents, and in many cases considerably less homology, for
example 75% or 80%, will be acceptable. Locating the parts of these
sequences that are not critical may be time consuming, but is
routine and well within the skill in the art. Exemplary techniques
for modifying oligonucleotide sequences include using
polynucleotide-mediated, site-directed mutagenesis. See Zoller et
al. (1984); Higuchi et al. (1988); Ho et al. (1989); Horton et al.
(1989); and PCR Technology: Principles and Applications for DNA
Amplification, (ed.) Erlich (1989).
[0151] The invention provides for a plant optimized sequence
encoding an immunoprotective antigen of interest. A plant-optimized
coding sequence is designed with hybrid codon preference reflecting
tomato and potato codon usage (Ausubel F., et al., eds. (1994)
Current Protocols in Molecular Biology, vol. 3, p. A.1C.3 Haq TA,
Mason H S, Clements J D, Arntzen C J (1995).
[0152] A plant optimized sequence of the invention can be prepared
as described in U.S. Pat. No. 5,380,831, incorporated by reference
herein in its entirety. In general, the frequency of codon usage
for a target plant of interest is used to adjust the codon usage
frequency of a target gene of interest, for example, NDV HN.
[0153] The native sequence is scanned for sequence motifs that
might result in interference with expression in the target plant,
such as poly-A addition sites, Shaw/Kamen degradation sites, splice
junction sites, and anything related to RNA termination or
potential hairpin formation, etc. Runs of A/T sequences are often
avoided. In one embodiment, it is preferable to keep strings of A/T
to four or fewer in a row, if possible, since most regulatory sites
tend to contain runs of A's and T's (e.g. AATAAA consensus poly-A
or ATTTA Shaw/Kamen). In general it is useful to scan for about 16
putative poly-A addition sequences based on identified sites from
plants found in the literature. In certain embodiments, it is
useful to search for C/G runs since they can stabilize hairpin stem
formation. Since monocots tend to favor third position C's and G's
somewhat more than dicots, the relevance of identification of C/G
runs may depend on the host plant target for expression.
[0154] In one embodiment wherein a gene is expressed in both dicots
and monocots, an overall plant codon usage frequency is used as a
basis for sequence optimization.
[0155] For the purposes of the present invention the term membrane
anchor sequence contemplates that which the ordinarily skilled
artisan understands about the term. Membrane anchor sequences
include transmembrane protein sequences and are found in many
naturally occurring proteins. Such membrane anchor sequences vary
in size but always are comprised of a series of amino acids with
lipophilic or aliphatic side chains that favor the hydrophobic
environment within the membrane. During RNA translation and post
translational processing, the anchor sequences integrate and become
embedded in the cell membrane and function to anchor, or loosely
attach the protein to a cellular membrane component allowing
hydrophilic portions of the protein to be exposed to, and interact
with, the aqueous milieu inside or outside of the cell.
[0156] In preparing the constructs of this invention, the various
DNA fragments may be manipulated, so as to provide for the DNA
sequences in the proper orientation and, as appropriate, in the
proper reading frame. Adapters or linkers may be employed for
joining the DNA fragments or other manipulations may be involved to
provide for convenient restriction sites, removal of superfluous
DNA, removal of restriction sites, or the like.
[0157] In carrying out the various steps, cloning is employed, so
as to amplify a vector containing the promoter/gene of interest for
subsequent introduction into the desired host cells. A wide variety
of cloning vectors are available, where the cloning vector includes
a replication system functional in E. coli and a marker which
allows for selection of the transformed cells. Illustrative vectors
include pBR322, pUC series, pACYC184, Bluescript series
(Stratagene) etc. Thus, the sequence may be inserted into the
vector at an appropriate restriction site(s), the resulting plasmid
used to transform the E. coli host (e.g., E. coli strains HB101,
JM101 and DH5.alpha.), the E. coli grown in an appropriate nutrient
medium and the cells harvested and lysed and the plasmid recovered.
Analysis may involve sequence analysis, restriction analysis,
electrophoresis, or the like. After each manipulation the DNA
sequence to be used in the final construct may be restricted and
joined to the next sequence, where each of the partial constructs
may be cloned in the same or different plasmids.
[0158] Vectors are available or can be readily prepared for
transformation of plant cells. In general, plasmid or viral vectors
should contain all the DNA control sequences necessary for both
maintenance and expression of a heterologous DNA sequence in a
given host. Such control sequences generally include a leader
sequence and a DNA sequence coding for translation start-signal
codon, a translation terminator codon, and a DNA sequence coding
for a 3' UTR signal controlling messenger RNA processing. Selection
of appropriate elements to optimize expression in any particular
species is a matter of ordinary skill in the art utilizing the
teachings of this disclosure. Finally, the vectors should desirably
have a marker gene that is capable of providing a phenotypical
property which allows for identification of host cells containing
the vector.
[0159] The activity of the foreign coding sequence inserted into
plant cells is dependent upon the influence of endogenous plant DNA
adjacent to the insert. Generally, the insertion of heterologous
genes appears to be random using any transformation technique;
however, technology currently exists for producing plants with site
specific recombination of DNA into plant cells (see WO 91/09957).
Any method or combination of methods resulting in the expression of
the desired sequence or sequences under the control of the promoter
is acceptable.
[0160] The present invention is not limited to any particular
method for transforming plant cells. Technology for introducing DNA
into plant cells is well-known to those of skill in the art. Four
basic methods for delivering foreign DNA into plant cells have been
described. Chemical methods (Graham and van der Eb, Virology,
54(02):536-539, 1973; Zatloukal, Wagner, Cotten, Phillips, Plank,
Steinlein, Curiel, Bimstiel, Ann. N.Y. Acad. Sci., 660:136-153,
1992); Physical methods including microinjection (Capecchi, Cell,
22(2):479-488, 1980), electroporation (Wong and Neumann, Biochim.
Biophys. Res. Conmmun. 107(2):584-587, 1982; Fromm, Taylor, Walbot,
Proc. Natl. Acad. Sci. USA, 82(17):5824-5828, 1985; U.S. Pat. No.
5,384,253) and the gene gun (Johnston and Tang, Methods Cell.
Biol., 43(A):353-365, 1994; Fynan, Webster, Fuller, Haynes,
Santoro, Robinson, Proc. Natl. Acad. Sci. USA 90(24):11478-11482,
1993); Viral methods (Clapp, Clin. Perinatol., 20(1):155-168, 1993;
Lu, Xiao, Clapp, Li, Broxmeyer, J. Exp. Med. 178(6):2089-2096,
1993; Eglitis and Anderson, Biotechniques, 6(7):608-614, 1988;
Eglitis, Kantoff, Kohn, Karson, Moen, Lothrop, Blaese, Anderson,
Avd. Exp. Med. Biol., 241:19-27, 1988); and Receptor-mediated
methods (Curiel, Agarwal, Wagner, Cotten, Proc. Natl. Acad. Sci.
USA, 88(19):8850-8854, 1991; Curiel, Wagner, Cotten, Bimstiel,
Agarwal, Li, Loechel, Hu, Hum. Gen. Ther., 3(2):147-154, 1992;
Wagner et al., Proc. Natl. Acad. Sci. USA, 89, (13):6099-6103,
1992).
[0161] The introduction of DNA into plant cells by means of
electroporation is well-known to those of skill in the art. Plant
cell wall-degrading enzymes, such as pectin-degrading enzymes, are
used to render the recipient cells more susceptible to
transformation by electroporation than untreated cells. To effect
transformation by electroporation one may employ either friable
tissues such as a suspension culture of cells, or embryogenic
callus, or immature embryos or other organized tissues directly. It
is generally necessary to partially degrade the cell walls of the
target plant material to pectin-degrading enzymes or mechanically
wounding in a controlled manner. Such treated plant material is
ready to receive foreign DNA by electroporation.
[0162] Another method for delivering foreign transforming DNA to
plant cells is by microprojectile bombardment. In this method,
microparticles are coated with foreign DNA and delivered into cells
by a propelling force. Such micro particles are typically made of
tungsten, gold, platinum, and similar metals. An advantage of
microprojectile bombardment is that neither the isolation of
protoplasts (Cristou et al., 1988, Plant Physiol., 87:671-674) nor
the susceptibility to Agrobacterium infection is required. An
illustrative embodiment of a method for delivering DNA into maize
cells by acceleration is a Biolistics Particle Delivery System,
which can be used to propel particles coated with DNA or cells
through a screen onto a filter surface covered with corn cells
cultured in suspension. The screen disperses the particles so that
they are not delivered to the recipient cells in large aggregates.
For the bombardment, cells in suspension are preferably
concentrated on filters or solid culture medium. Alternatively,
immature embryos or other target cells may be arranged on solid
culture medium. The cells to be bombarded are positioned at an
appropriate distance below the macroprojectile stopping plate. In
bombardment transformation, one may optimize the prebombardment
culturing conditions and the bombardment parameters to yield the
maximum numbers of stable transformants. Both the physical and
biological parameters for bombardment are important in this
technology. Physical factors are those that involve manipulating
the DNA/microprojectile precipitate or those that affect the flight
and velocity of the microprojectiles. Biological factors include
all steps involved in manipulation of cells before and immediately
after bombardment, the osmotic adjustment of target cells to help
alleviate the trauma associated with bombardment, and also the
nature of the transforming DNA, such as linearized DNA or intact
supercoiled plasmids.
[0163] Agrobacterium-mediated transfer is a widely applicable
system for introducing foreign DNA into plant cells because the DNA
can be introduced into whole plant tissues, eliminating the need to
regenerate an intact plant from a protoplast. The use of
Agrobacterium-mediated plant integrating vectors to introduce DNA
into plant cells is well known in the art. See, for example, the
methods described in Fraley et al., 1985, Biotechnology, 3:629;
Rogers et al., 1987, Meth. in Enzymol., 153:253-277. Further, the
integration of the Ti-DNA is a relatively precise process resulting
in few rearrangements. The region of DNA to be transferred is
defined by the border sequences, and intervening DNA is usually
inserted into the plant genome as described in Spielmann et al.,
1986, Mol. Gen. Genet., 205:34; Jorgensen et al., 1987, Mol. Gen.
Genet., 207:471.
[0164] Modern Agrobacterium transformation vectors are capable of
replication in E. coli as well as Agrobacterium, allowing for
convenient manipulations. Moreover, recent technological advances
in vectors for Agrobacterium-mediated gene transfer have improved
the arrangement of genes and restriction sites in the vectors to
facilitate construction of vectors capable of expressing various
proteins or polypeptides. Convenient multi-linker regions flanked
by a promoter and a polyadenylation site for direct expression of
inserted polypeptide coding genes are suitable for present
purposes. In addition, Agrobacterium containing both armed and
disarmed Ti genes can be used for the transformations.
[0165] Transformation of plant protoplasts can be achieved using
methods based on calcium phosphate precipitation, polyethylene
glycol treatment, electroporation, and combinations of these
treatments (see, e.g., Potrykus et al., 1985, Mol. Gen. Genet.,
199:183; Marcotte et al., Nature, 335:454, 1988). Application of
these systems to different plant species depends on the ability to
regenerate the particular species from protoplasts.
[0166] Once the plant cells have been transformed, selected and
checked for antigen expression, it is possible in some cases to
regenerate whole fertile plants. This will greatly depend on the
plant species chosen. Methods for regenerating numerous plant
species have been reported in the literature and are well known to
the skilled artisan. For practice of the present invention, it is
preferable to transform plant cell lines that can be cultured and
scaled-up rapidly by avoiding the generally lengthy regeneration
step. In addition the use of plant cell cultures avoids open field
production and greatly reduces the chances of gene escape and food
contamination. Tobacco suspension cell cultures such as NT-1 and
BY-2 (An, G., 1985 Plant Physiol. 79, 568-570) are preferred
because these lines are particularly susceptible to handling in
culture, are readily transformed, produce stably integrated events
and are amenable to cryopreservation.
[0167] The tobacco suspension cell line, NT-1, is suitable for the
practice of the present invention. NT-1 cells were originally
developed from Nicotiana tabacum L.cv. bright yellow 2. The NT-1
cell line is widely used and readily available; though, any tobacco
suspension cell line is consistent with the practice of the
invention. It is worth noting that the origins of the NT-1 cell
line are unclear. Moreover, the cell line appears variable and is
prone to change in response to culture conditions. NT-1 cells
suitable for use in the examples below are available from the
American Type Culture Collection under accession number ATCC No.
74840. See also U.S. Pat. No. 6,140,075, herein incorporated by
reference.
[0168] Many plant cell culture techniques and systems ranging from
laboratory-scale shaker flasks to multi-thousand liter bioreactor
vessels have been described and are well know in the art of plant
cell culture. See for example Fischer, R. et al, 1999 Biotechnol.
Appl. Biochem. 30, 109-112 and Doran, P., 2000 Current Opinions in
Biotechnology 11, 199-204. After the transformed plant cells have
been cultured to the mass desired, they are harvested, gently
washed and placed in a suitable buffer for sonication. Many
different buffers are compatible with the present invention. In
general the buffer is an aqueous isotonic buffered salt solution at
or near a neutral pH value that does not contain any detergent.
Preferred buffers include Dulbeccos Phosphate Buffered Saline and
PBS containing 1 mM EDTA.
[0169] For sonication, the washed cells are placed in buffer in a
range of about 0.01 gm/ml to about 5.0 gm/ml, preferably in a range
of about 0.1 gm/ml to about 0.5 gm/ml (washed wet weight cells per
volume of buffer). Many commercially available sonication
instruments are consistent with the invention and sonication times
range from about 5 to about 20 seconds, preferably about 15 to
about 20 seconds. The resulting particles are membrane vesicles
that may range in size from a few microns to several hundred
microns and expose the recombinant, immunoprotective, anchored
proteins.
[0170] An immunoprotective agent or antigen of interest is
expressed and isolated according to methods well known in the art
and described in the examples herein below.
[0171] In one embodiment, a method of producing an antigen of
interest comprises preparing a transgenic plant comprising a vector
encoding the antigen. The plant is incubated under conditions
wherein the plant expresses the antigen prior to the onset of
ripening of the plant. According to this embodiment, the plant has
a fruit that ripens (including but not limited to tomato, banana,
citrus, melon, strawberry, pineapple, stonefruit, mango, pumpkin,
squash etc.) The antigen produced according to this method can be
isolated from the plant, or from the fruit of the plant prior to
administration. Alternatively, the antigen is not isolated from the
plant but is administed in a crude, food-processed or raw form. The
details of this method are described in the Examples below.
Plants Useful According to the Invention
[0172] The present invention also provides for a transgenic plant
transformed with the constructs of the invention. Plants that can
be used for practice of the present invention include any
dicotyledon and monocotyledon. These include, but are not limited
to, tobacco, tomato, potato, eggplant, pepino, yam, soybean, pea,
sugar beet, lettuce, bell pepper, celery, carrot, asparagus, onion,
grapevine, muskmelon, strawberry, rice, sunflower, rapeseed/canola,
wheat, oats, maize, cotton, walnut, spruce/conifer, poplar and
apple, berries such as strawberries, raspberries, alfalfa and
banana. Since many edible plants used by humans for food or as
components of animal feed are dicotyledenous plants, dicotyledons
are typically employed, although monocotyledon transformation is
also applicable especially in the production of certain grains
useful for animal feed. It is particularly advantageous in certain
disease prevention for human infants to produce a vaccine in a
juice for ease of administration to humans such as juice of tomato,
soybean, and carrot, or milk. Cells and seeds derived from these
plant vaccines are also useful according to the invention.
[0173] Representative plants that have been transformed with this
system and representative references are listed in Table A. Other
plants having edible parts, or which can be processed to afford
isolated protein, can be transformed by the same methods or routine
modifications thereof.
TABLE-US-00001 TABLE A Plant Reference Tobacco Barton, K. et al.,
(1983) Cell 32, 1033 Tomato Fillatti, J. et al., (1987)
Bio/Technology 5, 726-730 Potato Hoekema, A. et al., (1989)
Bio/Technology 7: 273-278 Eggplant Filipponee, E. et al., (1989)
Plant Cell Rep. 8: 370-373 Pepino Atkinson, R. et al., (1991) Plant
Cell Rep. 10: 208-212 Yam Shafer, W. et al., (1987) Nature. 327:
529-532 Soybean Delzer, B., et al., (1990) Crop Sci. 30: 320-322
Pea Hobbs, S. et al., (1989) Plant Cell Rep. 8: 274-277 Sugar beet
Kallerhoff, J. et al., (1990) Plant Cell Rep. 9: 224-228 Lettuce
Michelmore, R., et al., (1987) Plant Cell Rep. 6: 439-442 Bell
pepper Liu, W. et al., (1990) Plant Cell Rep. 9: 360-364 Celery
Liu, C-N. et al., (1992) Plant Mol. Biol. 1071-1087 Carrot Liu,
C-N. et al, (1992) Plant Mol Biol. 1071-1087 Asparagus Delbriel, B.
et al., (1993) Plant Cell Rep. 12: 129-132 Onion Dommisse, E. et
al.; (1990) Plant Sci. 69: 249-257 Grapevine Baribault, T., et al.,
(1989) Plant Cell Rep. 8: 137-140 Muskmelon Fang, G., et al.,
(1990) Plant Cell Rep. 9: 160-164 Strawberry Nehra, N. et al.,
(1990) Plant Cell Rep. 9: 10-13 Rice Raineri, D. et al., (1990)
Bio/Technology. 8: 33-38 Sunflower Schrammeijer, B. et al., (1990)
Plant Cell Rep. 9: 55-60 Rapeseed/ Pua, E. et al., (1987)
Bio/Technology 5. 815 Canola Wheat Mooney, P. et al., (1991) Plant
Cell Tiss. Organ Cult. 25: 209-218 Oats Donson, J. et al., (1988)
Virology. 162: 248-250 Maize Gould, J. et al., (1991) Plant
Physiol. 95: 426-434 Alfalfa Chabaud, M. et al., (1988) Plant Cell
Rep. 7: 512-516 Cotton Umbeck, P. et al., (1987) Bio/Technology. 5:
263-266 Walnut McGranahan, G. et al., (1990) Plant Cell Rep. 8:
512-516 Spruce/Conifer Ellis, D. et al., (1989) Plant Cell Rep. 8:
16-20 Poplar Python, F. et al., (1987) Bio/Technology 5: 1323 Apple
James, P. et al., (1989) Plant Cell Rep. 7: 658-661
[0174] A transgenic plant transformed with a vector described
hereinabove is another aspect of the present invention.
[0175] Potato varieties FL 1607 ("Frito Lay 1607") and Desiree, and
tomato variety Tanksley TA234TM2R are particularly preferred
varieties, which have been transformed with binary vectors using
the methods described herein. Of these transformed varieties,
Desiree is the only commercial variety; the other varieties can be
obtained from Frito-Lay (Rhinelander, Wis.) and Steve Tanksley
(Dept. of Plant Breeding, Cornell Univ.). Potato variety FL1607
allows rapid transformation but is not a good agronomic variety as
it suffers from hollow heart.
[0176] Tomato is preferred as a model system for expression of
foreign proteins because of its ease of genetic transformation, and
because fruit-specific, ripening dependent promoters are available
for regulated expression (Giovannoni et al., 1989).
[0177] The invention includes whole plants, plant cells, plant
organs, plant tissues, plant seeds, protoplasts, callus, cell
cultures, and any group of plant cells organized into structural
and/or functional units capable of expressing at least a
polynucleotide of the invention. Preferably, whole plants, plant
cells, plant organs, plant tissues, plant seeds, protoplasts,
callus, cell cultures, and any group of plant cells produce 0.001,
0.01, 1, 5, 10, 25, 50, 100, 500, or 11000 .mu.g of polypeptide of
the invention per gram of total soluble plant material.
Use, Dosage and Administration of a Vaccine According to the
Invention
[0178] Food plant produced antigens provide a less expensive source
of antigen, that does not require animal-sourced components, for
the preparation of vaccines.
[0179] The vaccines according to the invention are useful for
protection against a pathogen of interest and against viral
infection.
[0180] 1. Administration
[0181] The invention provides for methods of administering a
vaccine according to the invention to a mammal to prevent viral
infection.
[0182] In one embodiment, a vaccine is administered orally (either
by feeding or by oral gavage) to ensure inducing a mucosal immune
response as well as to take advantage of cost and convenience.
Conveniently, an oral administration step entails consuming a
transgenic plant or plant part according to the invention. An
edible vaccine according to the invention can be in the form of a
plant part, an extract, a juice, a liquid, a powder or a
tablet.
[0183] An vaccine according to the invention may also be
administered by via an intranasal route in the form of a nasal
spray. Alternatively, a vaccine according to the invention may be
administered orally, intraperitoneally, intramuscularly,
intravenously, or subcutaneously.
[0184] The invention provides for compositions comprising an edible
vaccine admixed with a physiologically compatible carrier. As used
herein, "physiologically compatible carrier" refers to a
physiologically acceptable diluent such as water, phosphate
buffered saline, or saline, and further may include an adjuvant.
Adjuvants such as incomplete Freund's adjuvant, aluminum phosphate,
aluminum hydroxide, or alum are materials well known in the
art.
[0185] The invention also provides for pharmaceutical compositions.
In addition to the active ingredients, these pharmaceutical
compositions may contain suitable pharmaceutically acceptable
carrier preparations which can be used pharmaceutically.
[0186] Pharmaceutical compositions for oral administration can be
formulated using pharmaceutically acceptable carriers well known in
the art in dosages suitable for oral administration. Such carriers
enable the pharmaceutical compositions to be formulated as tablets,
pills, dragees, capsules, liquids, gels, syrups, slurries,
suspensions and the like, for ingestion by the patient.
[0187] Pharmaceutical preparations for oral use can be obtained
through combination of active compounds with solid excipient,
optionally grinding a resulting mixture, and processing the mixture
of granules, after adding suitable auxiliaries, if desired, to
obtain tablets or dragee cores. Suitable excipients are
carbohydrate or protein fillers such as sugars, including lactose,
sucrose, mannitol, or sorbitol; starch from corn, wheat, rice,
potato, or other plants; cellulose such as methyl cellulose,
hydroxypropylmethyl-cellulose, or sodium carboxymethyl cellulose;
and gums including arabic and tragacanth; and proteins such as
gelatin and collagen. If desired, disintegrating or solubilizing
agents may be added, such as the cross-linked polyvinyl
pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium
alginate.
[0188] Dragee cores are provided with suitable coatings such as
concentrated sugar solutions, which may also contain gum arabic,
talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol,
and/or titanium dioxide, lacquer solutions, and suitable organic
solvents or solvent mixtures. Dyestuffs or pigments may be added to
the tablets or dragee coatings for product identification or to
characterize the quantity of active compound, i.e., dosage.
[0189] Pharmaceutical preparations which can be used orally include
push-fit capsules made of gelatin, as well as soft, sealed capsules
made of gelatin and a coating such as glycerol or sorbitol.
Push-fit capsules can contain active ingredients mixed with a
filler or binders such as lactose or starches, lubricants such as
talc or magnesium stearate, and, optionally, stabilizers. In soft
capsules, the active compounds may be dissolved or suspended in
suitable liquids, such as fatty oils, liquid paraffin, or liquid
polyethylene glycol with or without stabilizers.
[0190] Pharmaceutical formulations for parenteral administration
include aqueous solutions of active compounds. For injection, the
pharmaceutical compositions of the invention may be formulated in
aqueous solutions, preferably in physiologically compatible buffers
such as Hank's solution, Ringer's solution, or physiologically
buffered saline. Aqueous injection suspensions may contain
substances which increase the viscosity of the suspension, such as
sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally,
suspensions of the active solvents or vehicles include fatty oils
such as sesame oil, or synthetic fatty acid esters, such as ethyl
oleate or triglycerides, or liposomes. Optionally, the suspension
may also contain suitable stabilizers or agents which increase the
solubility of the compounds to allow for the preparation of highly
concentrated solutions.
[0191] For nasal administration, penetrants appropriate to the
particular barrier to be permeated are used in the formulation.
Such penetrants are generally known in the art.
[0192] 2. Manufacture and Storage
[0193] The pharmaceutical compositions of the present invention may
be manufactured in a manner known in the art, e.g. by means of
conventional mixing, dissolving, granulating, dragee-making,
levitating, emulsifying, encapsulating, entrapping or lyophilizing
processes.
[0194] The pharmaceutical composition may be provided as a salt and
can be formed with many acids, including but not limited to
hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic,
etc . . . Salts tend to be more soluble in aqueous or other
protonic solvents that are the corresponding free base forms. In
other cases, the preferred preparation may be a lyophilized powder
in 1 mM-50 mM histidine, 0.1%-2% sucrose, 2%-7% mannitol at a pH
range of 4.5 to 5.5 that is combined with buffer prior to use.
[0195] After pharmaceutical compositions comprising a compound of
the invention formulated in an acceptable carrier have been
prepared, they can be placed in an appropriate container and
labeled for treatment of an indicated condition with information
including amount, frequency and method of administration.
[0196] 3. Therapeutically Effective Dose
[0197] Pharmaceutical compositions suitable for use in the present
invention include compositions wherein the active ingredients are
contained in an effective amount to achieve the intended purpose.
The determination of an effective dose is well within the
capability of those skilled in the art.
[0198] For any compound, the therapeutically effective dose can be
estimated initially either in cell culture assays, or in animal
models, usually birds, mice, rabbits, dogs, or pigs. The animal
model is also used to achieve a desirable concentration range and
route of administration. Such information can then be use to
determine useful doses and routes for administration in humans.
[0199] A therapeutically effective dose refers to that amount of
protein or its antibodies, antagonists, or inhibitors which prevent
or ameliorate the symptoms or conditions, for example caused by
viral infection. Therapeutic efficacy and toxicity of such
compounds can be determined by standard pharmaceutical procedures
in cell cultures or experimental animals, eg, ED50 (the dose
therapeutically effective in 50% of the population) and LD50 (the
dose lethal to 50% of the population). The dose ratio between
therapeutic and toxic effects is the therapeutic index, and it can
be expressed as the ratio, LD50/ED50. Pharmaceutical compositions
which exhibit large therapeutic indices are preferred. The data
obtained from cell culture assays and animals studies is used in
formulating a range of dosage for human use. The dosage of such
compounds lies preferably within a range of circulating
concentrations that include the ED50 with little or no toxicity.
The dosage varies within this range depending upon the dosage from
employed, sensitivity of the patient, and the route of
administration.
[0200] The exact dosage is chosen by the individual physician or
veterinarian in view of the patient to be treated. Dosage and
administration are adjusted to provide sufficient levels of the
active moiety or to maintain the desired effect. Additional factors
which may be taken into account include the severity of the disease
state; age, weight and gender of the subject; diet, time and
frequency of administration, drug combination(s), reaction
sensitivities, and tolerance/response to therapy. Long acting
pharmaceutical compositions might be administered every 3 to 4
days, every week, or once every two weeks depending on a half-life
and clearance rate of the particular formulation.
[0201] In general, compositions contain from about 0.5% to about
50% of the compounds in total, depending on the desired doses and
the type of composition to be used. The amount of the compounds,
however, is best defined as the effective amount, that is, the
amount of each compound which provides the desired dose to the
subject in need of such treatment. The activity of the adjunctive
combinations does not depend on the nature of the composition, so
the composition is chosen and formulated solely for convenience and
economy. Any of the combinations may be formulated in any desired
form of composition.
[0202] Dosage amounts may vary from 0.1 to 100,000 micrograms of
recombinant protein; transformed plant cell, or transformed
transgenic plant per subject per day, for example, 1 ug, 10 ug, 100
ug, 500 ug, 1 mg, 10 mg, and even up to a total dose of about 1 g
per subject per day, depending upon the route of administration. In
one embodiment, the dosage is in the range of 1 ng to) 0.5 mg per
kilogram bodyweight. In another embodiment, the dosage is in the
range of 1 .mu.g to 50 .mu.g per kilogram bodyweight. In another
embodiment, the dosage is in the range of 1 to 25 .mu.g per
kilogram bodyweight. In another embodiment, the dosage is in the
range of 2 to 25 .mu.g per kg body weight. In another embodiment,
the dosage is in the range of 2 to 15 .mu.g per kg bodyweight. For
example, in one embodiment HN antigen is administered
subcutaneously in a range of 2.5 to 5 .mu.g, and IN/ocularly in a
range of 0.5 to 12 .mu.g; HA antigen is administered subcutaneously
at a dose of 1 to 5 mg, IN/ocuraly in a range of 24 to 26 .mu.g;
VP2 antigen is administered subcutaneously in a range of 8 to 17
.mu.g, and LT antigen is administered orally in a range of 50 to
100 ng, subcutaneously in a range of 2-10 .mu.g and IN/ocularly in
a range of 2 to 10 .mu.g; Guidance as to particular dosages and
methods of delivery is provided in the literature. See U.S. Pat.
Nos. 4,657,760; 5,206,344; or 5,225,212, hereby incorporated by
reference. Those skilled in the art will employ different
formulations for nucleotides than for proteins or their inhibitors.
Similarly, delivery of polynucleotide or polypeptides will be
specific to particular cells, conditions, locations, etc.
Testing the Efficacy of a Vaccine of the Invention
[0203] The efficacy of a vaccine according to the invention is
determined by demonstrating that the administration of the vaccine
prevents or ameliorates the symptoms of the viral infection being
treated or prevented or the symptoms induced by the pathogen of
interest, by at least 5%, preferably 10-20% and more preferably,
25-100%.
[0204] The efficacy of a vaccine according to the invention is
determined by measuring antibody production in response to
vaccination with a plant derived protein of interest, detection of
the production of antibody in response to vaccination with a plant
derived protein of interest, wherein the antibody inhibits
hemagluttination, and assessing the mortality of a subject that has
been inoculated and then challenged with a vaccine comprising an
immunoprotective antigen of the invention (all as described
hereinbelow).
[0205] Having now generally described the invention, the same will
be more readily understood through reference to the following
Examples which are provided by way of illustration, and are not
intended to be limiting of the present invention, unless
specified.
EXAMPLE 1
Vectors
[0206] Gene Construction The coding sequence of the HN gene of NDV
strain "Lasota" (GenBank accession AF077761) was analyzed for codon
use and the presence of undesired sequence motifs that could
mediate spurious mRNA processing and instability, or methylation of
genomic DNA. See Adang M J, Brody M S, Cardineau G, Eagan N, Roush
R T, Shewmaker C K, Jones A, Oakes J V, McBride K E (1993) The
construction and expression of Bacillus thuringiensis cryIIIA gene
in protoplasts and potato plants. Plant Mol Biol 21:1131-1145. A
plant-optimized coding sequence was designed with hybrid codon
preference reflecting tomato and potato codon usage (Ausubel F., et
al., eds. (1994) Current Protocols in Molecular Biology, vol. 3, p.
A.1C.3 Haq TA, Mason H S, Clements J D, Arntzen C J (1995) Oral
immunization with a recombinant bacterial antigen produced in
transgenic plants. Science 268:714-716). The designed sequence is
shown in FIG. 1. The synthetic HN gene was assembled by a
commercial supplier (Retrogen) and was received in two separate
plasmids containing either the 5' (p4187-4203-1) or 3'
(p42111-4235-ic-1) half of the gene cloned into pCR-Blunt.
[0207] Plasmid construction: Binary vectors for
Agrobacterium-mediated plant transformations were constructed based
on vector pBBV-PHAS-iaaH shown in FIG. 2, which uses the plant
selection marker phosphinothricin acetyl transferase (PAT),
described in U.S. Pat. Nos. 5,879,903; 5,637,489; 5,276,268; and
5,273,894 herein incorporated by reference, driven by the
constitutive cassaya vein mosaic virus promoter (CsVMV) described
in WO 97/48819. The iaaH gene and the phaseolin promoter sequence
were deleted by digestion of pBBV-PHAS-iaaH with PacI and religated
to form pCVMV-PAT; then the single HindIII site was deleted by
filling it with Klenow enzyme and religating to form pCP!H. The
CsVMV promoter was end-tailored by PCR using primers CVM-Asc
(5'-ATGGCGCGCCAGAAGGTAATTATCCAAG SEQ ID NO:5) and CVM-Xho
(5'-ATCTCGAGCCATGGTTTGGATCCA SEQ ID NO:6) on template pCP!H, and
the product was cloned in EcoRV-digested, T-tailed pBluescriptKS to
make pKS-CVM7. A map of pCP!H is shown in FIG. 3. The HN expression
cassette pKS-CHN was constructed by ligating the vector
pKS-CVM7/NcoI-EcoRI with 3 insert fragments: the HN 5' half on
NcoI/PstI, the HN 3' half on PstI/KpnI, and the soybean vspB 3'
element on KpnI-EcoRI (Haq 1995). The binary T-DNA vector pCHN was
then assembled by ligation of the vector pCP!H/AscI-EcoRI and the
AscI-EcoRI fragment of pKS-CHN. A map of pCHN is shown in FIG.
4.
[0208] The granule bound starch synthase (GBSS) promoter, described
in U.S. Pat. No. 5,824,798 herein incorporated by reference, was
used to make other vectors. These constructs were made using a
promoter fragment amplified from genomic DNA of Solanum tuberosum
L. cv. "Desiree" using primers designed from the sequence in
GenBank accession X83220 for the Chinese potato cultivar
"Dongnong". A mutagenic primer "GSS-Nco"
(5'-tgccatggtgatgtgtggtctacaa SEQ ID NO:7) was used to create a Nco
I site overlapping the translation initiation codon, along with
forward primer "GSS-1.8F" (5'-gatctgacaagtcaagaaaattg SEQ ID NO:8)
complimentary to the 5' region at -1800 bp; the 1825 bp PCR product
was cloned in T-tailed pBluescriptKS to make pKS-GBN, and
sequenced. A mutagenic primer "GSS-Xho" (5'-agctcGAGCTGTGTGAGTGAGTG
SEQ ID NO:9) was used to create a XhoI site just 3' of the
transcription start site along with primer "GSS-1.8F"; the 1550 bp
PCR product was cloned in T-tailed pBluescriptKS to make pKS-GBX,
and sequenced.
[0209] A GBSS promoter expression cassette containing the TEV 5'UTR
(untranslated region), described in U.S. Pat. No. 5,891,665 herein
incorporated by reference, was assembled by ligation of vector
pTH210 digested with HindIII/XhoI with the HindIII/XhoI fragment of
pKS-GBX, which effected a substitution of the CaMV 35S promoter
with the 811 bp GBSS promoter, to make pTH252A. See Haq TA, Mason H
S, Clements J D, Arntzen C J (1995) Oral immunization with a
recombinant bacterial antigen produced in transgenic plants.
Science 268:714-716. The HN gene was inserted into
pTH252A/NcoI-KpnI by ligation with the HN 5' half on NcoI/PstI and
the HN 3' half on PstI/KpnI to make pHN252A. The binary T-DNA
vector pgHN was made by ligation of the vector pGLTB (shown in FIG.
11) digested with NsiI and EcoRI with the fragments
pHN252A/NsiI-KpnI and pTH210/KpnI-EcoRI. A map of pgHN is shown in
FIG. 5.
[0210] A GBSS promoter expression cassette containing the GBSS
5'UTR, described in U.S. Pat. No. 5,824,798 herein incorporated by
reference, with its intron, was assembled by ligation of vector
pTH210 (Haq 1995) digested with HindIII/NcoI with the HindIII/NcoI
fragment of pKS-GBN, which effected a substitution of the
(cauliflower mosaic virus) CaMV 35S promoter/TEV 5'UTR with the
1084 bp GBSS promoter/5'-UTR, to make pTH251 A. The binary T-DNA
vectorpgHN151 was madeby ligation of the vectorpCLT105 (shown in
FIG. 12) with fragments pTH251A/HindIII-NcoI and pHN252A/NcoI-KpnI.
A map of pgHN151 is shown in FIG. 6.
[0211] A GBSS promoter expression cassette containing the GBSS
5'UTR with its intron and the bean phaseolin 3' element (described
in U.S. Pat. Nos. 5,270,200; 6,184,437; 6,320,101, herein
incorporated by reference) was constructed. First, pCP!H was
digested at the unique KpnI site, blunted with T4 DNA polymerase,
and religated to make pCP!HK, which has the KpnI site removed.
pCP!HK was digested with NsiI, followed by blunting with T4 DNA
polymerase, and then digestion with PacI. The resulting vector was
ligated with a 2848 bp fragment from pgHN151 digested with SacI,
followed by blunting with T4 DNA polymerase, and then digestion
with PacI, to make pgHN153. A map of pgHN153 is shown in FIG.
7.
[0212] A chimeric constitutive promoter (4OCS.DELTA.MAS U.S. Pat.
Nos. 5,001,060; 5,573,932 and 5,290,924 herein incorporated by
reference) was used to construct another expression vector for HN.
Plasmid, pAGM149, was digested with EcoRV and partial digestion
with BamHI. This fragment was ligated with pCHN digested with
PmeI/PstI and the 5' half of the synthetic HN gene obtained by
digestion of pKS-CHN with BamHI/PstI. The resulting pMHN is shown
in FIG. 8.
[0213] A plasmid containing the HA gene of AIV
A/turkey/Wisconsin/68 (H5N9) was obtained from David Suarez (SEPRL,
Athens, Ga.). It was end-tailored by PCR to add restriction sites
NcoI at the 5' and KpnI at the 3' end, and inserted into the vector
pIBT210.1 (Haq et al., 1995), containing the 35S promoter, TEV
5'-UTR, and vspB 3' end. The expression cassette was transferred to
the binary vector pGPTV-Kan (Becker et al., Plant Mol Biol 1992;
20: 1195-7) by digestion with HindIII and EcoRI (partial), to make
pIBT-HAO. The HA gene/vspB3' end fragment from pIBT-HAO was
obtained by digestion with NcoI and EcoRI (partial), and inserted
into pKS-CVM7 to make pKS-CHA. The cassette containing the CsVMV
promoter, HA gene, and vspB3' end was obtained from pKS-CHA by
digestion with AscI and EcoRI (partial), and ligated with pCP!H to
make pCHA, shown in FIG. 9.
[0214] A dicot expression vector containing the plant-optimized
nucleotide sequence of NDV-HN was constructed. The completed
construct contained the gene cassette; Arabidopsis thaliana (At)
Ubiquitin 3 (Ubi3) promoter v2/Newcastle Disease Virus Hemagluttin
Neuraminidase (NDV-HN)/vspb 3'UTR:: Cassaya Vein Mosaic Virus
(CsVMV) promoter/PAT selectable marker/Arabidopsis thaliana (At)
ORF 25 3'UTR in a binary expression vector.
[0215] The expression cassette was assembled by completing a 3-way
ligation (FIG. 48). The binary vector pCGUS was modified by
removing the CsVMV promoter and GUS gene. A restriction enzyme
digest with the enzymes HinDIII and KpnI (New England Biolabs)
liberated a DNA fragment of 8310 bp. The NDV-HN gene was isolated
from the plasmid pCHN as an NcoI/KpnI (New England Biolabs)
restriction enzyme digestion DNA fragment of 1731 bp. Finally, the
AtUbi3 promoter v2 was isolated from pDAB7121 as an NcoI/HindIII
(New England Biolabs) restriction enzyme digestion. The resulting
reaction produced a DNA fragment of 1732 bp. The DNA of all three
enzyme digestions was excised from agarose gel via the "QiaexII Gel
Extraction Kit" (Qiagen). A 3-way ligation was completed using
equimolar concentrations of all three DNA fragments. The ligation
was catalyzed by the "T4 DNA Ligase" (New England Biolabs). The
resulting ligation product was transformed into "One Shot Top10
Chemically Competent E. coli." (Invitrogen). Two colonies were
isolated from this transformation. Initial screening via
restriction enzyme digestion indicated that both clones produced
the expected DNA banding pattern. The restriction enzyme reactions
that were completed used the following enzymes; EcoRV, FspI,
HinDIII, NcoI, SacI, ScaI (New England Biolabs). Further
confirmation of the correct construct involved a sequencing
reaction over the AtUbi 3' promoter v2/NDV-HN border. A sequencing
reaction with the primer pUHN2 (tgg ttg gag cct agg gta ct) was
completed using the "Beckman CEQ Quick Start Kit" (Beckman
Coulter). The results of this sequencing reaction indicated that
the AtUbi3' promoter v2 DNA fragment did ligate with the NDV-HN DNA
fragment at the intended NcoI restriction site. Sequencing across
the NDV-HN/pCGUS border and pCGUS/AtUbi3' promoter v2 border
required additional steps. A PCR reaction of both borders was
completed. The NDV-HN/pCGUS border and pCGUS/AtUbi3' promoter v2
border were PCR amplified using the "FailSafe PCR Kit" (Epicenter).
Two reactions for the NDV-HN/pCGUS border were completed using the
FailSafe buffer's B and C with the PCR primers KpnI 5' (act aat act
taa tga taa ca) and KpnI 3' (ata cac tac ctc cac atg tt). The PCR
reactions for the pCGUS/AtUbi3' promoter v2 border were completed
using FailSafe buffer's B and C with the PCR primers HinDIII 5'
(tgccggttttcaggtaac ata) and HinDIII 3' (agt tag gcc cga ata gtt
tga a). All of the PCR reactions produced DNA fragments of the
expected length (.about.600 bp). The PCR amplifications of the
border regions were cloned into the "TOPO TA cloning kit with
pCR2.1-TOPO" (Invitrogen). Clones containing the amplified border
region were identified via an EcoRI restriction enzyme digestion
(New England Biolabs). To confirm that the intended ligation at
these border junctions did occur, a sequencing reaction was
completed using the "Beckman CEQ Quick Start Kit" (Beckman Coulter)
with the M13 reverse sequencing primer (aac agc tat gac cat g). The
results of these sequencing reactions indicated that the correct
ligation reaction did occur at the pCGUS/NDV-HN and the pCGUS/At
Ubi 3 promoter v2 borders.
EXAMPLE 2
Preparation of Transgenic Nicotiana tabacum
[0216] Three to 4 days prior to transformation, a 1 week old NT-1
culture was sub-cultured to fresh medium by adding 2 ml of the NT-1
culture into 40 ml NT-1 media. The sub-culture was maintained in
the dark at 25.+-.1.degree. C. on a shaker at 100 rpm.
NT-1 Medium
TABLE-US-00002 [0217] Reagent Per liter MS salts 4.3 g MES stock
(20X) 50 ml B1 inositol stock (100X) 10 ml Miller's I stock 3 ml
2,4-D (1 mg/ml) 2.21 ml Sucrose 30 g pH to 5.7 .+-. 0.03
[0218] B1 Inositol Stock (100.times.)(1 liter) [0219] Thiamine HCl
(Vit B1)-0.1 g [0220] MES (20.times.) (1 liter) [0221] MES
(2-N-morpholinoethanesulfonic acid)-10 g [0222] Myoinositol-10 g
[0223] Miller's I (1 liter) [0224] KH.sub.2PO.sub.4-60 g
[0225] Agrobacterium tumefaciens containing the expression vector
of interest was streaked from a glycerol stock onto a plate of LB
medium containing 50 mg/l spectinomycin. The bacterial culture was
incubated in the dark at 30.degree. C. for 24 to 48 hours. One
well-formed colony was selected, and transferred to 3 ml of YM
medium containing 50 mg/L spectinomycin. The liquid culture was
incubated in the dark at 30.degree. C. in an incubator shaker at
250 rpm until the OD.sub.600 was 0.5-0.6. This took approximately
24 hrs.
LB Medium
TABLE-US-00003 [0226] Reagent Per liter Bacto-tryptone 10 g Yeast
extract 5 g NaCl 10 g Difco Bacto Agar 15 g
YM Medium
TABLE-US-00004 [0227] Reagent Per liter Yeast extract 400 mg
Mannitol 10 g NaCl 100 mg MgSO.sub.4.cndot.7H.sub.20 200 mg
KH.sub.2PO.sub.4 500 mg
[0228] (Alternatively, YM in powder form can be purchased (Gibco
BRL; catalog #10090-011). To make liquid culture medium, add 11.1 g
to 1 liter water.)
[0229] On the day of transformation, 1 .mu.l of 20 mM
acetosyringone was added per ml of NT-1 culture. The acetosyringone
stock was made in ethanol the day of the transformation. The NT-1
cells were wounded to increase the transformation efficiency. For
wounding, the suspension culture was drawn up and down repeatedly
(20 times) through a 10 ml wide-bore sterile pipette. Four
milliliters of the suspension was transferred into each of 10,
60.times.15 mm Petri plates. One plate was set aside to be used as
a non-transformed control. Approximately, 50 to 100 .mu.l of
Agrobacterium suspension was added to each of the remaining 9
plates. The plates were wrapped with parafilm then incubated in the
dark on a shaker at 100 rpm at 25.+-.1.degree. C. for 3 days.
[0230] Cells were transferred to a sterile, 50 ml conical
centrifuge tube, and brought up to a final volume of 45 ml with NTC
medium (NT-1 medium containing 500 mg/L carbenicillin, added after
autoclaving). They were mixed, then centrifuged at 1000 rpm for 10
min in a centrifuge equipped with a swinging bucket rotor. The
supernatant was removed, and the resultant pellet was resuspended
in 45 ml of NTC. The wash was repeated. The suspension was
centrifuged, the supernatant was discarded, and the pellet was
resuspended in 40 ml NTC. Aliquots of 5 ml were plated onto each
Petri plate (150.times.15 mm) containing NTCB10 medium (NTC medium
solidified with 8 g/l Agar/Agar; supplemented with 10 mg/l
bialaphos, added after autoclaving). Plates were wrapped with
parafilm then maintained in the dark at 25.degree..+-.1.degree. C.
Before transferring to the culture room, plates were left open in
the laminar flow hood to allow excess liquid to evaporate. After 6
to 8 weeks, putative transformants appeared. They were selected and
transferred to fresh NTCB5 (NTC medium solidified with 8 g/l
Agar/Agar; supplemented with 5 mg/l bialaphos, added after
autoclaving). The plates were wrapped with parafilm and cultured in
the dark at 25.degree..+-.1.degree. C.
[0231] Putative transformants appeared as small clusters of callus
on a background of dead, non-transformed cells. These calli were
transferred to NTCB5 medium and allowed to grow for several weeks.
Portions of each putative transformant were selected for ELISA
analysis. After at least 2 series of analysis by ELISA, lines with
the highest antigen levels were selected. The amount of callus
material for each of the elite lines was then multiplied in plate
cultures and occasionally in liquid cultures. The resulting
transformed NT-1 cell lines expressed and accumulated the HN
protein from Newcastle Disease Virus (Lasota strain), or
transformed cell line CHA expressed the HA protein from Avian
Influenza Virus. These lines contain an undetermined number of
copies of the T-DNA region of the plasmids stably integrated into
the nuclear chromosomal DNA. The transgenic CHN NT-1 cells
accumulate HN at levels up to 1% of total soluble protein as
determined by HN-specific ELISA.
[0232] Transgenic NT1 cell and potato lines selected for
Bialaphos.RTM. resistance were propagated and evaluated for HN
expression by ELISA. High-expressing NT1 cell lines were
established in liquid suspension culture. Potato lines containing a
constitutive promoter construct (pCHN, pMHN) were screened for
expression in leaf tissue, and selected lines were transferred to
soil and cultured in a greenhouse to obtain tubers for evaluation.
Potato lines containing a tuber-specific GBSS promoter construct
(PGHN, pGHN151, pGHN153) were screened for expression using
microtubers developed in vitro.
[0233] HN expression in pCHN-transformed NT1 lines using the CsVMV
promoter. Expression of HN in NT1 cell lines assayed from callus
growing on solid media is shown in FIG. 19. The highest expressing
lines were CHN-5 (8.5 ng/.mu.g TSP) and CHN-18 (6.2 ng/.mu.g TSP).
Lines CHN-1 and CHN-5 were established in liquid suspension
culture. The expression of HN per unit cell mass in these cultures
is shown in FIG. 20. Line CHN-5 showed expression of HN at 6.7
.mu.g per g cell mass. The same cell lines shown in FIG. 20 were
evaluated multiple times, and some new lines assayed at the last
time point, stability of expression of HN in the NT1 lines (FIG.
21). Western blotting of extracts from lines CHN-5 and CHN-7 showed
a single reactive band co-migrating with the reference standard
when probed with monoclonal antibody, and showed an additional
smaller band when probed with a polyclonal antiserum (FIG. 22).
[0234] Effects of freeze-drying of fresh cells and of storage of
extracts at 4.degree. C. In order to examine the effect of drying
on antigen stability, freeze-dried NT1 cells were extracted and
assayed by ELISA. Extracts of freeze-dried cells showed no loss or
apparent increase in HN content per fresh cell mass (FIG. 23).
Furthermore, extracts of fresh cells stored at 4.degree. C. for one
week showed an increase in HN content assayed by ELISA (FIG. 23).
We have observed a similar effect with another membrane-bound viral
protein, the hepatitis B surface antigen. It may result from
oxidation of cysteine residues to form correct disulfide bonds,
which results in display of the appropriate antigenic epitopes.
[0235] Particle behavior of plant-expressed HN antigen. In order to
evaluate assembly of NT1 cell-expressed antigen to form particulate
structures, sucrose gradient sedimentation was performed on crude
cell extracts. The profiles shown in FIG. 24 indicate that the NT1
cell-derived HN showed 2 peaks of ELISA reactive material. One peak
co-sedimented with inactivated virus particles, while the other
peak sedimented more slowly but still showed particulate character.
These data provide evidence that the HN protein is correctly
inserted into the ER membrane.
[0236] HN expression in pMHN-transformed NT1 lines using the
(4-ocs).DELTA.MAS promoter. HN expression in several NT1 cell lines
transformed with pMHN, compared to pCHN-transformed NT1 cell lines,
is shown in FIG. 25. Expression in pMHN-transformed lines was at
least as high as in pCHN-transformed lines, with the highest
accumulation of HN observed at approximately 30 .mu.g per gram cell
mass. Maximal HN expression in pCHN-transformed NT1 cells was less
than 20 .mu.g per gram cell mass. Bialaphos.RTM. resistant NT1 cell
lines were also generated and assayed for HA expression by ELISA as
described previously. In the first set of assays, only one
pCHA-transformed line accumulated HA to a similar extent as the
pGPTV-HAO line #12 that was previously generated (FIG. 13). In this
experiment, the expression range was up to 2.5 ng/.mu.g TSP. In
repeated experiments with these and new pCHA-transformed lines,
accumulation of HA ranged up to 18 ng/.mu.g TSP in line CHA-13
(FIG. 14). Selected lines from this group were analyzed by Western
blot. In all pCHA-lines tested, a reactive band at the expected
size of .about.68 kDa was observed (FIG. 15). These data show that
the HA protein was correctly processed to remove the signal
peptide, and accumulated in a stable form. Previous studies on
pGPTV-HAO-transformed NT1 cells by non-denaturing Western blot
(unpublished studies), showed that the HA assembled oligomeric
structures, probably the native trimer that occurs on the surface
of the AIV virion.
EXAMPLE 3
Cryopreservation
[0237] Cell Culture: NT-1 tobacco suspension cultures
(non-transgenic and transgenic lines) were maintained in 250 ml
Erlenmeyer flasks. Initially, the cells were cultured in a modified
liquid Linsmaier and Skoog medium (LS) (1965). The medium,
designated LSg, contained LS salts and vitamins, 30 g 1.sup.-1
glucose, and 0.05 mg 1.sup.-1 2,4-dichlorophenoxyacetic acid
(2,4-D). The pH of the medium was adjusted to 5.8. The cultures
were transferred to fresh medium weekly by transferring 6 ml of
7-day-old cultures into 50 ml of LSg medium.
[0238] Based on poor growth of the cells in LSg medium, two
additional media were investigated which were designated KCMS and
NT-1. KCMS contained Murashige and Skoog (MS) (1962) salts, 1.3 mg
1.sup.-1 thiamine, 200 mg 1.sup.-1 KH.sub.2PO.sub.4, 30 g 1.sup.-1
sucrose, 0.2 mg 1.sup.-1 and 0.1 mg 1.sup.-1 kinetin. NT-1 medium
contained MS salts, 180 mg 1.sup.-1 KH.sub.2PO.sub.4, 0.5 mg
1.sup.-1 2-N-morpholinoethanesulfonic acid, 1 mg 1.sup.-1 thiamine,
100 mg 1.sup.-1 myoinositol, 30 g 1.sup.-1 sucrose, and 2.21 mg
1.sup.-1 2,4-D. The pH of both media was adjusted to 5.7. For
transfers to fresh medium, 2 ml of a 7-day-old culture were
transferred into 48 ml of either KCMS or NT-1. All suspension
cultures were maintained in the dark at 25.degree. C. on an orbital
shaker at 100 rpm
[0239] Preculture: Three days after subculture (late exponential
growth period), the cells were precultured in their respective
medium (LSg, KCMS, or NT-1) by replacing one third of the medium
with 1M mannitol (for final concentration of 0.3M), for 24 to 72
h.
[0240] Heat Shock Treatment: After preculture, cultures were placed
on an orbital shaker at 100 rpm at 37.degree. C. for 2 h. They were
transferred back to the shaker at 25.degree. C. for 4 h before
vitrification.
[0241] Vitrification: The vitrification solution designated
PVS2/100% contained 30% glycerol, 15% ethylene glycol, and 15% DMSO
in a 0.4 M sucrose solution. A PVS2/20% solution was made by
diluting PVS2/100%. Both solutions were adjusted to a pH of 5.8,
autoclaved, then stored at 4.degree. C.
[0242] To start the vitrification process, 4 ml of ice cold
PVS2/20% was added to a 1 ml settled cell volume of cells. The
preparation was incubated on ice for 5 min, then 1 ml of cold
PVS2/100% was added at 1-minute intervals until a total volume of 9
ml was achieved. The preparations were centrifuged for 1 min at
7500-8000 rpm. The supernatant was discarded, then 0.5 ml of
PVS2/100% was added at 2,1-min intervals while the cells remained
on ice. Then 1 ml of PVS2/100% was added 3 times at 1-min
intervals. Half a milliliter of this mixture was then transferred
into each of 6 cryogenic straws (Continental Plastic Corporation,
Delavan, Wis.). The straws were heat sealed at each end with hot
forceps, and immersed immediately into liquid nitrogen. The
remaining 2 ml were not frozen and served as controls.
[0243] Recovery: After 1 h in liquid nitrogen, the vitrified cells
were thawed in a 40.degree. C. water bath for 3-5 sec, then
immediately diluted with 7 ml of cold 1.2 M sucrose. The cells were
kept on wet ice for 20 min, then centrifuged for 3 min at 7500-8000
rpm. Cells were transferred to 2 layers of filter paper (42.5 mm
Whatman) on LSg or NT-1 solidified medium containing 0.75% agarose
(Invitrogen Life Technologies, Carlsbad, Calif.) in 60.times.15 mm
tissue culture plates. The cultures were maintained at 25.degree.
C. in the dark. Two days after plating, the cells were transferred
to fresh NT-1 solidified medium. Transgenic lines were initially
plated on medium without a selection agent until cell growth was
well established and covered a small plate. They were then
transferred to medium containing the appropriate selection.
[0244] Thereafter, the cells were transferred to fresh medium
approximately every 2 wk. When cell growth nearly covered the
surface of the medium in the plate, the cells were removed from the
filter paper and transferred to larger plates (100.times.15 mm).
When the callus again covered the plate, the cells were transferred
to NT-1 medium solidified with 8 g 1.sup.-1 Agar (Sigma, St. Louis,
Mo.) for maintenance.
EXAMPLE 4
Antigen Preparation
[0245] Whole wet NT-1 cells expressing either HN, HA or null
control were harvested directly from cell culture and filtered to
remove excess media by placing a Spectramesh 30 filter in a Buchner
funnel and pouring cells and media through the filter using a
slight vacuum. 0.5 grams of cells were placed in 2 mls of buffer
(Dulbeccos Phosphate Buffered Saline and 1 mM EDTA), and then
sonicated for 15 to 20 seconds on ice. Sonication was performed
using a Branson 450 sonifier with a replaceable microtip at output
control of 8, duty cycle 60 for varying amounts of time. Sonicates
were then placed on ice until use.
EXAMPLE 5
Antigen Extraction
[0246] To examine whether non-detergent treatments could release
ELISA signal from transformed NT-1 cells and allow retention of
biological activity, a series of treatments were set up that
involved comparison of treatments without detergent and various
levels of sonication. The results were striking in that periods of
sonication greater than 20 seconds in extraction buffer completely
destroyed hemagglutination activity of HN from a pCHN bearing NT-1
cell line, but not ELISA signal. In contrast, sonication for only
20 seconds in DPBS not only released antigen detectable by ELISA
signal, but the soluble protein extracts demonstrated excellent
hemagglutination activity (see Table 1).
TABLE-US-00005 TABLE 1 Comparison of extraction methods on
hemagglutination activity of plant-derived HN Ext. buffer DPBS DPBS
Ext. buffer DPBS F/T F/T Sonic. Sonic. Sonic. Sonic. Sonic. Sample
1.5 min 1.5 min 15 sec 15 sec 15 sec pCHN-18-NT-1 .ltoreq.2 256
4096 1024 1024 pCHA-47-NT-1 .ltoreq.2 -- 64 16 16 NT-1 .ltoreq.2
.ltoreq.2 .ltoreq.2 .ltoreq.2 .ltoreq.2 Native NDV.sup.1 256 512
128 nd nd .sup.1Native NDV was sonicated for 2 minutes. Ext. buffer
- 50 mM sodium ascorbate, 1 mM EDTA, 1 mM PMSF, and 0.1% Triton
X-100 pH 7.2; DPBS--Dulbeccos phosphate buffered saline;
sonic.--sonication; F/T--freeze-thaw; nd--not done for this
experiment.
[0247] Plant-derived HN extracted without detergent was used as the
antigen in hemagglutination inhibition assays to determine if
polyclonal antibody produced to native virus could recognize and
inhibit agglutination of RBC's by the plant-derived HN. The results
indicate that native antibody will recognize the hemagglutination
epitope of the plant-derived HN in a similar manner as native
virus' (Table 2). The data from Table 2 also demonstrates that
control NT-1 cells or NT-1 cells expressing a non-hemagglutinating
protein do not agglutinate red blood cells nor are affected by NDV
specific serum. In this experiment, extracts of plant-derived
protein were diluted to 4 HA units, and then treated with NDV
specific polyclonal antisera. Four HA units is the standard amount
of virus used for titration of serum.
TABLE-US-00006 TABLE 2 Comparison of hemagglutination inhibition
(HAI) activity of plant-derived HN and native virus
Hemagglutination HN Inhibition Titer Concentration Hemagglutination
(chicken anti-NDV Sample ELISA Titer polyclonal antibody) NDV
allantoic 20 ug/ml 4* 4096 fluid (native) NT control cell None
.ltoreq.2 .ltoreq.8 pCHN-7-NT-1 1.5 ug/g fresh >64 512 weight
CHN-18-NT-1 12 ug/g fresh .gtoreq.4096 1024 weight CLT-101-14- None
.ltoreq.2 .ltoreq.8 NT-1 *Stock virus is diluted such that 4HA
units, a 1:4 dilution of the stock will generate a positive HA but
a dilution of 1:8 will not hemagglutinate. This is the
concentration of virus used to titer antibody, the endpoint
dilution of antibody that will interfere with 4 HA units of virus
is considered to be the HAI titer of the antibody preparation.
[0248] The above data demonstrates that using an extraction method
that does not utilize detergent and reduces the amount of cell
disruption produces an extracellular fraction that retains
hemagglutination activity for transformed NT-1 cell lines
expressing HN or HA. To determine if HN protein from non-detergent
extracted NT-1 cells had additional biological activity that may be
relevant to vaccine efficacy, the HN extracts were examined for
ability to bind to chicken cell receptors. immuno-fluorescence
staining indicated that CEF cells treated with native virus or
pCHN-18 extracts were indistinguishable. Thus, plant-derived HN
retains virus-like ability to bind to receptors on target cell
surfaces.
[0249] The combined data from Tables 1 and 2, together with the
hemagglutination and immunofluorescence assays discussed above,
suggest that the HN protein derived transgenic NT-1 cells will
retain both immunological and biological features if processed and
formulated correctly. Most significant of the data provided above
is that antisera to native virus will recognize plant-derived HN in
HAI tests. Chickens that contain at least 4 fold higher titer of
HAI activity above background are almost always certain of
protection against challenge from virulent virus. To test whether
the plant derived protein extracted in non-detergent as described
above would generate antibody in target animals species both HA and
HN protein were prepared and inoculated into chickens and
rabbits.
EXAMPLE 6
Quantitative ELISA
[0250] HN
[0251] Quantitative ELISA for HN is performed by coating plates on
the day prior to running the assay. 50 .mu.l per well of Capture
Antibody (Rabbit anti-HN in 50% glycerol, diluted (1:500) in 0.01M
Borate Buffer) is added to each well of each flat bottom 96-well
microtiter plate. The plate is covered and incubated at 2.degree.
C.-7.degree. C. overnight, (12-18 hours). The coated ELISA plate(s)
are allowed to equilibrate to room temperature (approximately 20-30
minutes) and then washed three times with 200-300 .mu.l per well
per wash with PBS-T. The entire plate is blocked to prevent
non-specific reactions by adding 200 .mu.l per well of 3% Skim Milk
Blocking Solution. The plate(s) is(are) then incubated for 2 hours
(+10 minutes) at 37.degree. C..+-.2.degree. C. (covered with a
plate cover or equivalent). HN Reference antigen (Ag) in 1% Skim
Milk Blocker is added to a concentration of 250 ng HN/ml;
experimental antigens are diluted in 1% Blocker. The HN ELISA
plate(s) are washed one time with PBS-T and 100 .mu.l per well of
diluted HN Reference Antigen and HN Test Samples are added to Row
B. 50 .mu.l per well of 1% Blocker is added to all remaining wells.
The samples are serially diluted down the plate by transferring 50
.mu.l per well from row B to row G, mixing 4-5 times with the
pipette before each transfer. The plate(s) are covered and
incubated 1 hour (+10 minutes) at 37.degree. C..+-.2.degree. C.;
and the ELISA plate(s) are washed three times with PBS-T. Fifty
.mu.l of NDV HN 4A Ascites Fluid in 50% glycerol (1:2000) in 3%
Blocker is added to each well and the plates are covered and
incubated 1 hour (+10 minutes) at 37.degree. C..+-.2.degree. C. The
ELISA plate(s) are washed three times with PBS-T and 50 .mu.l of
rabbit anti-Mouse IgG in 50% glycerol (1:3000) in 3% Blocker is
added to each well. The plates are covered and incubated 1 hour
(+10 minutes) at 37.degree. C..+-.2.degree. C. ELISA plate(s) are
washed three times with PBS-T and 50 .mu.l of ABTS Peroxidase
Substrate Solution (equilibrated at RT (room temperature) for at
least 30 minutes) is added to each well. The plates are covered and
incubated at RT in the dark for 15-20 minutes. The Optical Density
(OD) of the wells are read at a wavelength of 405 nm (with a 492 nm
Reference Filter). The initial dilution of the HN Reference Antigen
should be within 0.7-1.0 OD, this serves as the positive control
for the ELISA.
[0252] HA
[0253] For quantitative ELISA of HA, the plates are coated on the
day prior to running the assay. Fifty .mu.l per well of Capture
Antibody (goat anti-Hav5 in 50% glycerol, diluted (1:1000) in 0.01M
Borate Buffer) is added to each well of flat bottom 96-well
microtiter plate(s)). The plate(s) are covered and incubate at
2.degree. C.-7.degree. C. overnight, (12-18 hours). The coated
ELISA plate(s) is(are) allowed to equilibrate to room temperature
(approximately 20-30 minutes) and is(are) then washed three times
with 200-300 .mu.l per well per wash with PBS-T. The entire plate
is blocked to prevent non-specific reactions by adding 200 .mu.l
per well of 3% Skim Milk Blocking Solution. The plate(s) is(are)
then incubated for 2 hours (+10 minutes) at 37.degree.
C..+-.2.degree. C. (covered with a plate cover or equivalent).
AIV-HA (allanotoic fluid) reference Antigen is added in 1% Skim
Milk Blocker to a concentration of 1000 ng HA/ml and experimental
antigens are diluted in 1% Blocker. The HA ELISA plate(s) are
washed one time with PBS-T and 100 .mu.l per well of diluted HA
reference antigen and HA Test Samples are added to Row B. 50 .mu.l
per well of 1% Blocker is added to all remaining wells. The samples
are serially diluted down the plate by transferring 50 .mu.l per
well from row B to row G, mixing 4-5 times with the pipette before
each transfer. The plate(s) are covered and incubated 1 hour (+10
minutes) at 37.degree. C..+-.2.degree. C. The ELISA plate(s) are
washed three times with PBS-T. Fifty .mu.l of chicken anti-AIV
polyclonal antisera in 50% glycerol (1:2000) in 3% Blocker is added
to each well and the plates are covered and incubated 1 hour (+10
minutes) at 37.degree. C..+-.2.degree. C. The ELISA plate(s) are
washed three times with PBS-T and then 50 .mu.l of goat
anti-chicken IgG in 50% glycerol (1:3000) in 3% Blocker is added to
each well. The plates are covered and incubated 1 hour (+10
minutes) at 37.degree. C..+-.2.degree. C. The ELISA plate(s) are
washed three times with PBS-T and 50 .mu.l of ABTS Peroxidase
Substrate Solution (equilibrated at RT for at least 30 minutes) is
added to each well. The plate(s) are covered and incubated at RT in
the dark for 15-20 minutes. The Optical Density (OD) of the wells
read at a wavelength of 405 nm (with a 492 nm Reference Filter).
The initial dilution of the HA Reference Antigen should be within
0.7-1.0 OD, this serves as the positive control for the ELISA.
EXAMPLE 7
Serum ELISA
[0254] NDV-HN
[0255] Plates are coated with rabbit a-NDV pooled antiserum (Mixed
1:2 with 50% glycerol in water) diluted (1:2000) in 0.01 M borate
buffer (100 .mu.l/well). Plates are incubated overnight at
2-7.degree. C., covered and then equilibrated for approximately
20-30 minutes at room temperature. Plates are washed 3.times. with
PBS-T (1.times.PBS+0.05% Tween-20) at 300 .mu.l/well with the
Titertek M96 plate washer or equivalent. Plates are blocked with 5%
skim milk in PBS-T (Blocking Buffer) (200 .mu.l/well) and incubated
for 2 hours at 37.degree. C. Plates are washed 1.times. with PBS-T
at 300 .mu.l/well with the Titertek M96 plate washer or equivalent.
NDV allantoic fluid is diluted 1:200 in Blocking Buffer. 100
.mu.l/well of the diluted antigen is added to the plate, and plates
are incubated for 1 hour at 37.degree. C. Plates are washed
3.times. with PBS-T at 300 .mu.l/well with the Titertek M96 plate
washer or equivalent. Test chicken serum samples are diluted
(1:50). Negative control serum is diluted (1:50) (Neg. Control
27NOV00). Positive control serum is diluted (1:10,000 or 1:20,000)
(SPAFAS Chicken .alpha.-NDV serum). All serum samples are diluted
in Blocking Buffer. 100 .mu.l/well of Negative Control Serum is
added to Column 1 Rows B-G; 200 .mu.I/well of Positive Control
Serum is added to Columns 2-3 Row A; 200 .mu.l/well of Test Serum
Samples is added to Rows A appropriate columns. This allows 4
samples per plate with 8 dilutions per sample. 100 .mu.l/well of
Blocking Buffer is added to all remaining wells The Positive
Control Serum and the Test Serum Samples are serially two-fold
diluted down the plate. The samples are diluted down the plate from
Row A to Row H, discarding the remaining 100 .mu.l/well. Plates are
incubated for 1 hour at 37.degree. C. and then washed 3.times. with
PBS-T at 300 .mu.l/well with the Titertek M96 plate washer or
equivalent. The Goat .alpha.-Chicken IgG (H&L)-HRP is diluted
(1:3000) in Blocking Buffer. 100 .mu.l/well of the diluted
conjugate is added to each plate. Once the conjugate is added to
the plates, the ABTS substrate is equilibrated at RT in the dark.
Plates are incubated for 1 hour at 37.degree. C. and then washed
3.times. with PBS-T at 300 .mu.l/well using the Titertek M96 plate
washer or equivalent. 100 .mu.l/well of pre-warmed ABTS substrate
is added to each plate, waiting 2-3 minutes between plates. Plates
are read at dual wavelength 405/490 nm on the Tecan Sunrise plate
reader or equivalent when the first dilution of the positive
control reaches an absorbance of between 0.7 and 1.0.
[0256] AIV-HA
[0257] Plates are coated with Rabbit .alpha.-HA pooled antiserum
diluted (1:1000) in 0.01 M borate buffer and incubated overnight at
2-7.degree. C., covered. Plates are equilibrated for approximately
20-30 minutes at room temperature and then washed 3.times. with
PBS-T (PBS Stock+0.05% Tween-20) at 300 .mu.l/well using the
Titertek M96 plate washer or equivalent. The plates are blocked
with 5% skim milk in PBS-T (Blocking Buffer) (200 .mu.l/well) and
incubated for 1 hour at 37.degree. C. The plates are washed
1.times. with PBS-T at 300 .mu.l/well using the Titertek M96 plate
washer or equivalent. Inactivated TIW/68 AIV Allantoic Fluid is
diluted (1:100) in Blocking Buffer and 100 .mu.l/well of the
diluted antigen is added to the plate. Plates are incubated for 1
hour at 37.degree. C. The plates are washed 3.times. with PBS-T at
300 .mu.l/well using the Titertek M96 plate washer or equivalent.
Test chicken serum samples are diluted (1:50). Negative control
serum is diluted (1:50). Positive control serum is diluted
(1:25600) (USDA/SEPRL Chicken .alpha.-AIV (TIW/68 antiserum) All
serum is diluted in Blocking Buffer. 100 .mu.l/well of Negative
Control Serum is added to Column 1 Rows B-G; 200 .mu.l/well of
Positive Control Serum is added to Columns 2-3 Row A; 200
.mu.l/well of Test Serum Samples is added to Row A in appropriate
columns; 100 .mu.l/well of Blocking Buffer is added to all
remaining wells. The Positive Control Serum and the Test Serum
Samples are serially diluted two-fold down the plate, discarding
the remaining 100 .mu.l/well. The plates are incubated for 1 hour
at 37.degree. C. The plates are washed 3.times. with PBS-T (300
.mu.l/well) using the Titertek M96 plate washer or equivalent. Goat
a-Chicken IgG (H&L)-HRP is diluted (1:3000) in Blocking Buffer
and 100 .mu.l/well of the diluted conjugate is added to each plate.
Once the conjugate is added to the plates, the ABTS substrate is
equilibrated at RT in the dark. The plates are incubated for 1 hour
at 37.degree. C. and then washed 3.times. with PBS-T at 300
.mu.l/well using the Titertek M96 plate washer or equivalent. 100
.mu.l/well of equilibrated ABTS substrate is added to each plate,
leaving 2-3 minutes between plates. Plates are read at dual
wavelength 405/490 nm on the Tecan Sunrise plate reader or
equivalent when the first dilution of the positive control reaches
an absorbance of between 0.7 and 1.0.
EXAMPLE 8
Antigenicity in Rabbits
[0258] To test whether the plant derived protein extracted in
non-detergent as described above would generate antibody in target
animals species both HA and HN protein were prepared and inoculated
into rabbits. New Zealand White rabbits 3 months of age were
inoculated with HA-AW or HN-NDV according to the dose schedule
provided in Table 3. For the primary inoculation the antigen was
mixed with Complete Freund's Adjuvant (CFA) and Incomplete Freund's
Adjuvant was used for all booster inoculations. The antibody titers
induced by both proteins are provided in Table 4. The results
indicate that after two inoculations HAI antibody titers were
induced by both proteins. However, the titers of the AIV-HA
inoculated rabbits were higher than those induced by the NDV-HN
protein. This may be significant since the AW/HA protein had lower
overall biological activity (hemagglutination) per unit of AIV-HA
protein than NDV-HN (Table 3 column 4). This may indicate that the
AIV-HA protein derived from plants is more potent per unit of
protein than the NDV-HN protein considering that the quantitation
methods from both proteins are accurate. It also suggests that the
AIV-HA protein formulated in this manner would be immunogenic in
chickens.
TABLE-US-00007 TABLE 3 Dose levels for inoculation into rabbits
ELISA Hemagglu- Results BCA TSP.sup.1 tination Hemagglutination (ug
Results units per Sample Endpoint Titers protein/ml) (mg/ml) ug
protein NT .ltoreq.2 0.00 1.44 0 Control CHN-7 2048 13.53 2.48 3027
CHN-18 1024 9.21 4.10 2223 CHA-13 32 3.80 6.15 168 CHA-47 16 2.88
5.25 111 .sup.1All NT-1 samples were provided from non freeze-dried
material. BCA--bicinchoninic acid, primary component of Pierce
Chemical BCA protein assay kit; TSP--total soluble protein.
TABLE-US-00008 TABLE 4 Serology Results from NT-1 derived AIV-HA
and NDV-HN inoculated rabbits Treatment with Sample NDV HAI Titers
NDV ELISA Titers NDV-HN Number Pre-Bleed 6 week 8 week 10 week
Pre-Bleed 6 week 8 week 10 week Sup from NT Control Cells 2723
.ltoreq.8 .ltoreq.8 .ltoreq.8 .ltoreq.8 0 0 0 0 Sup from HN - 18
Cells 2724 .ltoreq.8 23 23 23 0 815 554 888 Sup from HN - 18 Cells
2725 .ltoreq.8 11 23 23 0 0 585 591 Sup from HN - 7 Cells 2726
.ltoreq.8 45 23 23 0 607 461 0 Sup from HN - 7 Cells 2727 .ltoreq.8
45 45 45 0 1396 2008 1270 Treatment with Sample AIV HAI Titers AIV
ELISA Titers AIV-HA Number Pre-Bleed 6 week 8 week 10 week
Pre-Bleed 6 week 8 week 10 week Sup from NT Control Cells 2723
.ltoreq.8 .ltoreq.8 .ltoreq.8 .ltoreq.8 <25 <25 <25 <25
Sup from HA - 13 Cells 2728 .ltoreq.8 362 362 362 <25 25600
25600 25600 Sup from HA - 13 Cells 2729 .ltoreq.8 181 181 11 <25
3200 3200 <25 Sup from HA - 47 Cells 2730 .ltoreq.8 181 362 724
<25 12800 25600 25600 Sup from HA - 47 Cells 2731 .ltoreq.8
.ltoreq.8 362 362 <25 50 12800 25600
[0259] To examine the efficacy of the plant derived antigens in
chickens the HN protein was inoculated into chickens that were 2
days of age and 10 days of age. The dose concentrations used for
these studies are provided in Table 5. All vaccine inoculum were
formulated with the soluble fraction of NT-1 cells grown 15-20 days
in shaker flasks at 25.degree. C. Adjuvant used in both trials was
MPL-TDM from Corixa, Inc. Intranasal groups were given MPL alone as
the adjuvant.
EXAMPLE 9
Challenge in Poultry
[0260] Two-day old SPF chicks were inoculated by various routes
using biologically active (hemagglutination positive) NDV-HN
protein derived from NT-1 with the amount of HN protein per
inoculation shown in Table 5. The serological and challenge results
of this trial are provided in Table 6. All control groups responded
as expected in that birds not receiving NDV-HN antigen in the
inoculum had 100% mortality, whereas, control birds receiving 20 ug
of native NDV by SQ had 100% survival. In the experimental
treatment groups there was 75% protection in group #3 (SQ
inoculation without adjuvant) and 80% protection in group #4 (SQ
inoculation with adjuvant). The remaining treatment groups, which
were inoculated by IN and oral routes, had 100% mortality. However,
in group 6 two birds had a delay in mortality, indicating that
these birds may have been sensitized to vaccination (see Table
9)
TABLE-US-00009 TABLE 5 Dose levels used per inoculation for poultry
trial ug HN/Bird Group Day 0 Day 14 Day 21 NT Control (SQ) 0 0 0
NDV All. Fluid (SQ) 20 20 20 HN Tobacco (SQ) 150 230 180 HN Tobacco
(IN) 6 14 14 HN Tobacco (OG) 114 282 136 HN Tobacco 114og + 700of*
282og/1400of* 136 + 2366* (OG + OF) Average 3590 5810 6025
hemagglutination units per ug HN *Dose based on wet weight
expression per mass cells mixed with feed; IN--intranasal;
SQ--subcutaneous; OG--oral gavage; OF--on feed mixtures.
TABLE-US-00010 TABLE 6 Serology and challenge results from poultry
trial NDV HAI titers NDV ELISA titers Sample Day Day Pre Post Day
Pre Post Surv. Treatment Number 14 21 Day 28 Chall. Chall. 28
Chall. Chall. Chall. 1. Control NT Cells 924 .ltoreq.8 .ltoreq.8
.ltoreq.8 .ltoreq.8 na 0 0 0 No SQ 1061 .ltoreq.8 .ltoreq.8
.ltoreq.8 .ltoreq.8 na 0 0 0 No 1073 .ltoreq.8 .ltoreq.8 .ltoreq.8
.ltoreq.8 na 0 0 0 No 1077 .ltoreq.8 .ltoreq.8 .ltoreq.8 .ltoreq.8
na 0 0 0 No 1081 .ltoreq.8 .ltoreq.8 .ltoreq.8 .ltoreq.8 na 0 0 0
No 2. NDV HN allantoic 1063 11 1448 2896 724 724 11956 9245 7294
Yes fluid SQ 1068 11 1448 1024 724 362 9216 7639 6122 Yes 1072 45
1448 1448 724 362 11592 7500 5937 Yes 1083 23 724 724 181 91 5697
4919 3011 Yes 1089 45 1024 1448 362 181 15181 7449 6085 Yes 3. NDV
HN tobacco 797 23 45 45 .ltoreq.8 2896 0 0 19036 Yes SQ 1066
.ltoreq.8 16 45 .ltoreq.8 724 450 0 15087 Yes 1085 .ltoreq.8
.ltoreq.8 23 .ltoreq.8 na 0 0 na No 1095 .ltoreq.8 23 45 .ltoreq.8
724 436 0 10043 Yes 4. ND V HN tobacco 1067 11 45 45 .ltoreq.8 181
592 0 4912 Yes MPL/TDM adjuvant 1080 .ltoreq.8 181 181 45 181 1911
871 5048 Yes SQ 1093 11 45 45 .ltoreq.8 .ltoreq.8 0 0 0 Yes 1094 11
91 91 11 11 747 199 0 Yes 1098 .ltoreq.8 23 45 .ltoreq.8 na 0 0 na
No 5. NDV HN tobacco 796 .ltoreq.8 .ltoreq.8 .ltoreq.8 .ltoreq.8 na
0 0 na No IN 925 .ltoreq.8 .ltoreq.8 .ltoreq.8 .ltoreq.8 na 0 0 na
No 1065 .ltoreq.8 .ltoreq.8 .ltoreq.8 .ltoreq.8 na 0 0 na No 1084
.ltoreq.8 .ltoreq.8 .ltoreq.8 .ltoreq.8 na 0 0 na No 1092 .ltoreq.8
.ltoreq.8 .ltoreq.8 .ltoreq.8 na 0 0 na No 6. NDV HN tobacco + 921
.ltoreq.8 .ltoreq.8 .ltoreq.8 .ltoreq.8 na 0 0 na No MPL adjuvant
923 .ltoreq.8 11 .ltoreq.8 .ltoreq.8 na 0 0 na No IN 1069 .ltoreq.8
.ltoreq.8 .ltoreq.8 .ltoreq.8 na 0 0 na No 1074 .ltoreq.8 11
.ltoreq.8 .ltoreq.8 na 0 0 na No 1088 .ltoreq.8 11 8 .ltoreq.8 na 0
0 na No 7. NDV HN tobacco 723 .ltoreq.8 .ltoreq.8 .ltoreq.8
.ltoreq.8 na 0 0 na No Oral gavage 1062 .ltoreq.8 .ltoreq.8
.ltoreq.8 .ltoreq.8 na 0 0 na No 1075 .ltoreq.8 8 .ltoreq.8
.ltoreq.8 na 0 0 na No 1079 .ltoreq.8 8 .ltoreq.8 .ltoreq.8 na 0 0
na No 1086 .ltoreq.8 .ltoreq.8 .ltoreq.8 .ltoreq.8 na 0 0 na No 8.
NDV HN tobacco + 1070 .ltoreq.8 .ltoreq.8 .ltoreq.8 .ltoreq.8 na 0
0 na No MPL/TDM adjuvant 1082 .ltoreq.8 .ltoreq.8 .ltoreq.8
.ltoreq.8 na 0 0 na No Oral gavage + On feed 1091 .ltoreq.8
.ltoreq.8 .ltoreq.8 .ltoreq.8 na 0 0 na No 1097 .ltoreq.8 .ltoreq.8
.ltoreq.8 .ltoreq.8 na 0 0 na No 1100 .ltoreq.8 .ltoreq.8 .ltoreq.8
.ltoreq.8 na 0 0 na No All birds receive 10.sup.2 EID.sub.50 Texas
GB strain of NDV. Birds were challenged 24 days post last
vaccination. Bird numbers bolded had a delayed onset to mortality
see Table 9.
[0261] In a subsequent trial, eighteen 10-day old SPF birds were
inoculated according to the schedule and dose amounts described in
Table 7. Results of this trial are shown in Table 8. One control
group (#3), a non-vaccinated non-challenged treatment was used to
show that the housing and facility had no adverse affects on
general health of the chickens. Control groups in this trial also
responded as expected. Since birds from both trials were challenged
at the same facility, treatment group #2 served as a positive
control for both poultry trials. In the remaining groups, all of
which were inoculated SQ with HN derived from NT-1 cells, there was
100% survival in group #7, 80% survival in each of groups 5 and 6,
and 60% survival in group 4 (see Table 8).
TABLE-US-00011 TABLE 7 Dose levels of antigen used per inoculation
ug HN/Bird (Subcutaneous) Group Day 0 Day 14 Day 21 NT Control 0 0
0 NDV All. Fluid 20 20 20 HN Tobacco 20 20 20 (Low Dose) HN Tobacco
150 100 100 (High Dose) Average hemagglutination 3590 2625 2625
units per ug HN
[0262] Conclusions: Using a procedure that provides recovery of
hemagglutination of HA and HN protein, preparations for these
plant-derived proteins were inoculated into two separate animal
species by subcutaneous (SQ) route to determine if antibody could
be induced that will inhibit hemagglutination (HAI antibody). In
one trial, HN protein was formulated to have high hemagglutination
activity to total soluble protein ratios. These materials were then
inoculated by SQ, intranasal (IN) and by oral routes. The results
indicate that both HA-AIV and HN-NDV protein derived from NT-1
cells will induce hemagglutination inhibition (HAI) antibody in
rabbits when formulated in this manner. In addition, HN-NDV derived
from NT-1 inoculated by SQ route in chickens will induce (HAI)
antibodies and protect against virulent NDV challenge.
[0263] The results from these trials indicate that the HN-NDV
protein derived from NT-1 cells is immunogenic in birds when
inoculated by SQ. In most cases birds protected from challenge had
a detectable HAI titer post challenge, however, there were
exceptions to this observation. Two birds (bird #1093 and #1047,
respectively) did not have a detectable HAI or ELISA titer after
challenge but survived challenge (Tables 6 and 8).
TABLE-US-00012 TABLE 8 Serology and challenge results NDV HAI titer
NDV ELISA titers Sample Pre Post Pre Post Surv. Treatment Number
Day 21 Day 28 Chall. Chall. Day 21 Day 28 Chall. Chall. Chall. 1.
Control allantoic 1026 .ltoreq.8 .ltoreq.8 .ltoreq.8 na 0 0 0 na No
fluid 1027 .ltoreq.8 .ltoreq.8 .ltoreq.8 na 0 0 0 na No 1028
.ltoreq.8 .ltoreq.8 .ltoreq.8 na 0 0 0 na No 1029 .ltoreq.8
.ltoreq.8 .ltoreq.8 na 0 0 0 na No 1030 .ltoreq.8 .ltoreq.8
.ltoreq.8 na 0 0 0 na No 2. NDV HN allantoic 1031 362 91 181 91
9177 6937 5533 3551 Yes fluid 1032 362 724 181 91 12393 16533 7909
6080 Yes 20 ug/dose 1033 724 362 181 181 8622 15291 6766 6362 Yes
1034 362 181 181 181 7875 10071 6487 5822 Yes 1035 724 724 181 181
9681 16133 7537 6539 Yes 3. Control tobacco 1036 .ltoreq.8 23
.ltoreq.8 .ltoreq.8 0 0 0 0 n/c 1037 .ltoreq.8 11 .ltoreq.8
.ltoreq.8 0 0 0 0 n/c 1038 .ltoreq.8 .ltoreq.8 .ltoreq.8 .ltoreq.8
0 0 0 0 n/c 1039 .ltoreq.8 .ltoreq.8 .ltoreq.8 .ltoreq.8 0 0 0 0
n/c 1040 .ltoreq.8 23 .ltoreq.8 .ltoreq.8 0 0 0 0 n/c 4. NDV HN
tobacco 1041 11 <8 .ltoreq.8 1448 0 0 0 14042 Yes 20 ug/dose
1042 23 11 .ltoreq.8 2896 0 0 0 19263 Yes 1043 32 11 .ltoreq.8 na 0
0 0 na No 1044 .ltoreq.8 11 .ltoreq.8 na 0 0 0 na No 1045 23 11
.ltoreq.8 1024 0 0 0 11770 Yes 5. NDV HN tobacco 1046 23 23
.ltoreq.8 1448 0 674 0 11243 Yes 20 ug/dose + 1047 23 23 .ltoreq.8
.ltoreq.8 0 963 0 0 Yes MPL/TDM 1048 23 23 .ltoreq.8 na 396 757 0
na No emulsion adjuvant 1049 45 23 .ltoreq.8 362 0 804 0 6239 Yes
1050 11 11 .ltoreq.8 1448 0 398 0 15948 Yes 6. NDV HN tobacco 1051
91 45 23 181 1096 1137 565 4547 Yes 250 ug/dose 1052 45 45
.ltoreq.8 181 1166 998 0 7376 Yes 1053 23 23 .ltoreq.8 2896 0 0 0
16712 Yes 1054 23 23 .ltoreq.8 na 646 838 0 na No 1055 45 45 23 91
705 563 448 4902 Yes 7. NDV HN tobacco 1056 45 45 11 23 746 948 174
926 Yes 250 ug/dose + 1057 45 23 11 724 556 892 0 11542 Yes MPL/TDM
1058 45 23 23 724 780 1588 630 9915 Yes emulsion adjuvant 1059 91
64 23 91 2004 3090 1016 4690 Yes 1060 45 45 11 181 916 1522 448
6620 Yes All birds received 10.sup.2 EID.sub.50 Texas GB strain of
NDV, except group 1, which received 10.sup.4 EID.sub.50 Texas GB
strain of NDV and group 3, which was the non-challenge control.
Birds were challenged 31 days post last vaccination. Bird numbers
bolded had a delayed onset to mortality see Table 9.
n/c--nonchallenged.
[0264] Typically, a high titer response after challenge is
indicative of a good sensitizing immunization, yet, it is not
unprecedented with native or recombinant derived NDV antigen that
birds will be protected without a detectable HAI or ELISA antibody
titer (Winterfield, R. W., Dhillon, A. S., and L. J. Alby, 1979.
Vaccination of Chickens against Newcastle Disease with Live and
Inactivated Newcastle Disease Virus. Poultry. Sci. 59: 240-246). It
is proposed that either a cellular immune response or a local
immune response is involved with providing immune protection in a
vaccinated bird where no humoral antibody response can be detected
(Agrawal, P. K. and D. L. Reynolds. 1991. Evaluation of the cell
mediated immune response of chickens vaccinated with Newcastle
disease virus as determined by the under-agarose
leukocyte-migration inhibition technique. Avian Dis. 35:
360-364).
[0265] In some cases, although there was a detectable titer at the
end of the vaccination schedule at day 28, there was no protection
to challenge. However, in all cases when this situation occurred
there was no detectable antibody titer before (on the day of
challenge) or after challenge indicating that these birds were not
sensitized effectively (compare Tables 4 and 6). Differences
observed between the two poultry trials may be attributed to the
fact that antigen used in the first trial had a much higher
biological activity per microgram of HN. Antigen used in this trial
had at least a 2 fold higher level of hemagglutination activity per
microgram of protein than antigen in the latter trial (compare
Tables 5 and 7). This may be one reason why birds treated with
non-adjuvanted antigen (group #3, Table 6) developed detectable
ELISA and HAI titers by day 28, whereas, birds treated with
non-adjuvanted antigen (group #4, Table 8) did not show ELISA
titers by day 28. Another difference which may have contributed to
the results is the age of the birds. Birds in the first trial were
vaccinated at day 2 of age, whereas, birds in the second trial were
vaccinated at day 10 of age.
TABLE-US-00013 TABLE 9 Death on days after challenge D D % Group #
Treatment Route Chall. D 1 D 2 D 3 D 4 D 5 D 6 D 7 D 8 D 9 10 11-14
Surv. 1-018 Control allantoic fluid SQ 10.sup.4 0 0 3 2 -- -- -- --
-- -- -- 0 EID.sub.50 2-018 NDV HN allantoic fluid - 20 ug/ SQ
10.sup.2 0 0 0 0 0 0 0 0 0 0 0 100 dose EID.sub.50 3-018 Control
tobacco SQ None 0 0 0 0 0 0 0 0 0 0 0 100 4-018 NDV HN tobacco
derived - 20 SQ 10.sup.2 0 0 0 0 1 0 0 0 1 -- -- 60 ug/dose
EID.sub.50 5-018 NDV HN tobacco derived - 20 SQ 10.sup.2 0 0 0 0 0
1 0 0 0 0 0 80 ug/dose + MPL/TDM EID.sub.50 Emulsion adjuvant 6-018
NDV HN tobacco derived 250 ug/ SQ 10.sup.2 0 0 0 0 0 0 1 0 0 0 0 80
dose EID.sub.50 7-018 NDV HN tobacco derived 250 ug/ SQ 10.sup.2 0
0 0 0 0 0 0 0 0 0 0 100 dose + MPL/TDM Emulsion EID.sub.50 adjuvant
1-016 Control tobacco SQ 10.sup.2 0 0 0 4 1 -- -- -- -- -- -- 0
EID.sub.50 2-016 NDV HN allantoic fluid SQ 10.sup.2 0 0 0 0 0 0 0 0
0 0 0 100 EID.sub.50 3-016 NDV HN tobacco derived SQ 10.sup.2 0 0 0
0 0 0 0 0 0 0 1 80 EID.sub.50 4-016 NDV HN tobacco derived + SQ
10.sup.2 0 0 0 0 0 0 1 0 0 0 0 80 MPL/TDM Emulsion adjuvant
EID.sub.50 5-016 NDV HN tobacco derived IN 10.sup.2 0 0 0 2 3 -- --
-- -- -- -- 0 EID.sub.50 6-016 NDV HN tobacco derived + MPL IN
10.sup.2 0 0 0 3 0 0 0 2 -- -- -- 0 adjuvant EID.sub.50 7-016 NDV
HN tobacco derived oral gav. 10.sup.2 0 0 0 3 2 -- -- -- -- -- -- 0
EID.sub.50 8-016 NDV HN tobacco derived + MPL/ oral 10.sup.2 0 0 0
2 3 -- -- -- -- -- -- 0 TDM adj EID.sub.50
[0266] Adjuvant also seems to be an important feature in
formulating the antigen in these trials. Although the adjuvant
effect was not evident when using higher doses of NT-1 derived
NDV-HN (compare groups 3 and 4, Table 6 with groups 6 and 7, Table
8), there was a clear adjuvant effect when using a low dose of
NDV-HN (compare groups 4 and 5, Table 8). In addition, although
there was 100% mortality in groups inoculated by intranasal route,
the group that received adjuvant had 2 birds (#923 and #1088) that
did not die until day 8, which was 3 days after all negative
control birds had succumbed to challenge (Table 9). The
significance of the delay to mortality in the intranasal group
receiving adjuvanted antigen is not significant, however, it is
interesting that birds #923 and #1088 were two birds with
detectable HAI antibody titers at day 21 of the trial and were
treated with less antigen per dose than the other treatment groups
(see Table 5).
[0267] It is clear from the data provided here that HN-NDV derived
from transformed NT-1 cells is efficacious against virulent
challenge to NDV. The immune response to the plant derived antigen
has several similarities to immune response to native antigen. 1)
Although the antibody titers are higher for native antigen
pre-challenge, a 20 ug dose inoculated SQ will provide protection
against challenge for both native and plant derived antigen. 2)
Antibody titers must have similar duration of response in that in
both studies challenge was performed 24 days and 31 days post
vaccination. 3) All birds producing a positive HAI antibody
response (above background) at the end of the vaccination schedule
and post challenge were protected from NDV associated
pathology.
[0268] No birds were protected from challenge when inoculated by
oral or intranasal route. In the case of the oral administered
birds, inoculums of 100 to 300 ug of HN-NDV soluble protein from
NT-1 cells along with 700 to 2400 ug of HN-NDV feed as whole wet
cells per inoculation did not elicit a detectable antibody titer
after three doses nor were birds protected from challenge. Because
the HN-NDV from NT-1 has definite binding capability to red blood
cells and to (CEF) chick embryo fibroblasts, the ability to bind to
native receptors did not supplement binding or delivery to antigen
sampling sites on the bird mucosal surface in this study.
[0269] The data provided here show that HN-NDV antigen derived from
transgenic NT-1 cell culture will protect against virulent
challenge when administered SQ. Furthermore, despite feeding
several milligrams of antigen in the oral treatment groups, no HAI
antibody was induced in the systemic compartment and no protection
against challenge was observed. Thus, as previous data have shown,
antigens that do not have a natural affinity or invasiveness for
the antigen presenting sites on the mucosal surface need to be
targeted to those sites with the aid of a protein that does
sensitize the mucosal surface.
EXAMPLE 10
Preparation and Analysis of Transgenic Potato
[0270] Binary vectors pCHN, pgHN, pMHN and pCHA were used to
transform potato (cv. Desiree) and transgenic tubers analysed for
expression of the recombinant HN antigen from NDV, or the
recombinant HA antigen from Avian Influenza Virus.
[0271] Plant material. In vitro plants of Solanum tuberosum cv
Desiree were provided by Dr. Steven Slack, Department of Plant
Pathology, Cornell University. For propagation, nodal segments were
transferred to a shoot propagation medium designated CM which
contained MS salts (Murashige and Skoog, 1962), 100 mg/l
myoinositol, 0.4 mg/l thiamine, 20 g/l sucrose, and 8 g/l Agar/Agar
(Sigma Chemical Co., St. Louis, Mo.; catalog #A-1296). The pH of
the medium was adjusted to pH 5.7 before the addition of the
Agar/Agar. One nodal explant was placed in each test tube and
maintained at 24.degree..+-.1.degree. C. under a photoperiod of 16
h (light)/8 h (dark) at 74 .mu.E m.sup.-2 s.sup.-1. The source of
light for these cultures and those described throughout this report
was from a mixture of cool and warm fluorescent bulbs (F40CW and
F40WW) (Philips Lighting Co., www.lighting.philips.com/index.htm).
Nodal explants were harvested and transferred to fresh medium every
6 weeks.
[0272] Agrobacterium preparation. Agrobacterium tumefaciens
containing the gene construct of interest was streaked from a
glycerol stock maintained at -80.degree. C. onto Petri plates of LB
medium which contained 10 g/l bacto tryptone, 5 g/l yeast extract,
10 g/l NaCl, 50 mg/l spectinomycin, and 15 g/l Difco Bacto Agar
(Difco Laboratories, Detroit, Mich.; catalog #DF 0140-01). Four
well-formed colonies were picked with a sterile pipet tip, then
added to 50 ml of YM medium (Gibco BRL cat.# 10090-011) containing
50 mg/l spectinomycin. Cultures were grown in a shaking incubator
at 28.degree. C. and 100 rpm for 24 hrs, or until the culture
reached an OD.sub.600 of 0.5-0.7. It takes approximately 24 hrs to
reach this OD. When the desired OD was reached, the cells were
centrifuged at 8000 rpm for 10 min at 20.degree. C. The pellet was
resuspended in MS liquid medium (MS salts, 2 mg/l glycine, 0.5 mg/l
nicotinic acid, 0.5 mg/l pyridoxine, 0.4 mg/l thiamine, 0.25 mg/l
folic acid, 0.05 mg/l d-biotin, 100 mg/l myoinositol, 30 g/l
sucrose, pH 5.6) at the same original volume as the YM selective
medium.
[0273] Infection. Stem internode segments 0.5-1 cm in length were
excised from six-week-old in vitro plants and inoculated the same
day. Approximately 100 internode explants were incubated per 50 ml
of inoculum for 10 min, agitating occasionally. After the
incubation, they were blotted onto sterile filter paper, then
transferred to medium designated CIM which contained MS salts, 2
mg/l glycine, 0.5 mg/l nicotinic acid, 0.5 mg/l pyridoxine, 0.4
mg/l thiamine, 0.25 mg/l folic acid, 0.05 mg/l D-biotin, 100 mg/l
myoinositol, 30 g/l sucrose (grade II; PhytoTechnology
Laboratories, Shawnee Mission, Kans.), 1 mg/l benzyladenine (BA), 2
mg/l naphthaleneacetic acid (NAA) (added after autoclaving), and 6
g/l Agar/Agar (PhytoTechnology Laboratories, Shawnee Mission,
Kans.). The pH of the medium was adjusted to 5.6 before the
addition of the Agar/Agar. One hundred explants were cultured per
100.times.20 mm Petri plates. All cultures were maintained at
24.degree..+-.1.degree. C. under a photoperiod of 16 h (light)/8 h
(dark) at 74 .mu.E m.sup.-2s.sup.-1.
[0274] Plant regeneration. After 48 hrs of cocultivation, the
explants were transferred to 3C5ZR bialaphos selective medium which
contained MS salts, 0.1 mg/l thiamine, 0.5 mg/l nicotinic acid, 0.5
mg/l pyridoxine, 100 mg/l myoinositol, 30 g/l sucrose, 0.5 mg/l
indole-3-acetic acid (IAA) (added after autoclaving), 3 mg/l zeatin
riboside (added after autoclaving), 500 mg/l carbenicillin (added
after autoclaving) (Agri-Bio, Miami, Fla.), 5 mg/l bialaphos (added
after autoclaving) (Duchefa, http://www.duchefa.coml/), and 8 g/l
Agar/Agar. The pH of the medium was adjusted to 5.9 before the
addition of the Agar/Agar. Twenty-five internode segments were
cultured per 100.times.20 mm Petri plate and the plates were sealed
with Nesco film (Karlan Research Products, Santa Rosa, CA).
Explants were transferred weekly for 1 month, then every 10-14
days. All cultures were maintained at 24.degree..+-.1.degree. C.
under a photoperiod of 16 h (light)/8 h (dark) at 74
.mu.M.sup.-2S.sup.-1.
[0275] When regenerants were approximately 0.5-1 cm in length, they
were excised and transferred to bialaphos selective rooting medium
which contained the same components as CM with the addition of 500
mg/l carbenicillin (added after autoclaving) and 5 mg/l bialaphos
(added after autoclaving). Five regenerants were cultured per GA7
Magenta box. Once the shoots rooted, the shoot tip from each plant
was transferred to CM in test tubes for maintenance.
[0276] Microtubers. Microtubers were induced on plant material for
an early indication of expression in tubers. This was especially
applicable for transgenic lines containing genes driven by the
tuber-specific promoter, GBSS. Nodal segments were placed on
microtuber medium which contained 1/2 strength MS salts, 5 mg/l
kinetin, 80 g/l sucrose, 0.25 mM ancymidol (added after
autoclaving), 9 g/l Agar/Agar. The pH of the medium was adjusted to
5.85 prior to the addition of the Agar/Agar. The cultures were
maintained in the dark at 18.degree. C. The microtubers were
analyzed by ELISA for antigen expression levels.
[0277] PCR analysis. Genomic DNA was isolated from leaves from
3-4-week-old putative transformants. Leaf samples were homogenized
at room temperature in 500 .mu.l of an extraction buffer containing
200 mM Tris HCl (pH 7.5), 250 mM NaCl, 25 mM EDTA, and 0.5% SDS.
They were allowed to remain at room temperature for 1 hr, then
centrifuged at 12,000 rpm for 5 min. The supernatant was removed to
a new tube, 500 .mu.l of isopropanol was added, then the samples
either remained at room temperature for 5-10 min, or were placed at
-20.degree. C. overnight. They were then centrifuged at 13,000 rpm
for 5 min, and the supernatant was discarded. The resultant pellet
was washed with 70% ethanol, dried, then resuspended in 100 .mu.l
of TE buffer.
[0278] The primer set was designed such that the forward primer was
in the CVMV promoter and the reverse primer (PAT R2) was in the PAT
gene which resulted in a product size of approximately 500 bp.
Amplified DNA fragments were run on a 1% agarose gel, stained with
ethidium bromide, and visualized under a UV light.
[0279] ELISA analysis of leaves. Leaf material was harvested into
tubes then placed on ice. Lines with the highest antigen levels
were selected for propagation, then transferred to the
greenhouse.
[0280] Greenhouse acclimation. Plants with well-formed root systems
were transferred to Jiffy 7 pots. The pots were placed in trays and
covered with plastic domes. After approximately 2 weeks, the domes
were removed. The plants were transferred to 3 gallon pots
containing Cornell soil mix when the roots systems had grown
through the mesh on the Jiffy 7 pots.
[0281] HN expression in pCHN-transformed potato plants. Potato
(Solanum tuberosum L. cv. Desiree) plants were transformed with
pCHN, and regenerated Bialaphos.RTM. resistant plants were screened
for expression of HN in leaves by ELISA. Several lines were
selected based on leaf expression (FIG. 26), propagated, and
transplanted to soil for greenhouse culture. At maturity, tubers
were harvested, extracted, and assayed for HN content by ELISA
(FIG. 26). HN accumulation varied among individual tubers from the
same line, but in general expression was correlated with the HN
content of leaves within each line (FIG. 26). The best expression
was observed in tubers of lines 6, 21, 27, and 34, with the highest
accumulation observed at .about.11 .mu.g HN per g fresh tuber
mass.
[0282] Particle behavior of potato tuber-expressed HN antigen. In
order to evaluate assembly of NT1 cell-expressed antigen to form
particulate structures, sucrose gradient sedimentation was
performed on pCHN-transformed potato tuber extracts. The profiles
shown in FIG. 27 indicate that the tuber-derived HN showed 2 peaks
of ELISA reactive material, similar to the NT1 cell-derived HN
shown in FIG. 24.
[0283] HN expression in potato plants transformed with pGHN and
pGHN151. Potato (Solanum tuberosum L. cv. Desiree) plants were
transformed with pGHN or pGHN151, and regenerated Bialaphos.RTM.
resistant plants were screened for expression of HN in microtubers
by ELISA. Several pGHN-transformed lines were selected based on
microtuber expression (FIG. 28), propagated, and transplanted to
soil for greenhouse culture. Transformation with pGHN151 was
relatively inefficient, resulting in only one line that showed
expression in microtubers (GHN151-6), which was transplanted to
soil for greenhouse culture. At maturity, tubers were harvested,
extracted, and assayed for HN content by ELISA (FIG. 28). HN
accumulation varied among individual tubers from the same line, but
in general expression was correlated with the HN content of
microtubers within each line (FIG. 29). The best expression was
observed in tubers of lines GHN-1, 30, 47, and 54, with the highest
accumulation observed at 40 .mu.g HN per g fresh tuber mass.
Expression in tubers of line GHN151-6 varied between 6 and 12 .mu.g
HN per g fresh tuber mass. It is possible that the
intron-containing GBSS promoter construct pGHN151 was unstable in
transgenic plants or in Agrobacterium, resulting in poor expression
with this construct.
[0284] ELISA analysis of tubers. Approximately 3-4 months after
plants were transferred to soil, assorted tissue were harvested for
analysis. FIG. 16 shows expression of HA in microtubers of pCHA
transformed microtubers, which ranged up to 700 ng/g fresh weight.
This is similar to the accumulation observed in
pGPTV-HAO-transformed tubers (HA gene driven by CaMV 35S promoter),
which was maximal at 1 ug/g fresh weight. Selected lines were
transplanted to soil and grown in the greenhouse. Leaves of
soil-grown plants were sampled and assayed by ELISA (FIG. 17).
Expression of HA in leaves was very poor (<0.025 ng/.mu.g TSP),
which is consistent with the earlier assays with leaves of tissue
culture plants. Tubers of mature plants were harvested, extracted,
and evaluated for HA expression by ELISA. Accumulation of HA in
tubers was maximally 500 ng/g fresh weight (FIG. 18). The
expression observed in microtubers produced in vitro was well
correlated with the expression in soil-grown tubers (FIGS. 16 and
18), thus the microtuber is a good model for expression of HA with
pCHA.
EXAMPLE 11
Preparation and Analysis of Transgenic Tomato
[0285] Binary vectors pCHN, pMHN, and pUHN were used to transform
tomato (variety TA234) and transgenic fruit and leaves analysed for
expression of the recombinant HN protein from Newcastle Disease
Virus.
[0286] Plant material. Seeds from a tomato line designated TA234
were used for transformations. TA234, originally known as Momor, is
a verticillium and tobacco mosaic virus resistant line derived from
the variety Moneymaker. Seeds were surface sterilized in 20%
Clorox, for 20 min, rinsed 3 times with sterile distilled water,
then cultured on 1/2 MSO medium (See below) in Magenta boxes. They
were maintained at 24.degree..+-.1.degree. C., under a photoperiod
of 16 h (light)/8 h (dark) at 74 .mu.m.sup.-2S1. The source of
light for these cultures and those described throughout this report
was a mixture of cool and warm fluorescent bulbs (F40CW and F40WW)
(Philips Lighting Co., www.ligting.philips.com/index.htm). The seed
cultures were maintained for 6-8 days depending upon the stage of
cotyledon growth. Cotyledons were excised before the first true
leaves emerged. If cotyledon sections were longer than 1 cm, they
were cut into two 0.5 cm segments. Cotyledon sections were placed
on feeder layer plates which were prepared one day prior to
transformation. The feeder layer consisted of NT1 suspension
cultured cells plated on KCMS medium (See below) which had been
subcultured (2 mls of cells:48 ml of liquid KCMS) 7 days prior to
plating. The plated suspension culture was covered with a sterile 7
cm Whatman filter paper. Cotyledon sections were placed on top of
the filter paper.
[0287] Agrobacterium preparation. Agrobacterium tumefaciens
containing the gene construct of interest was streaked from
glycerol stocks maintained at -80.degree. C. onto fresh plates of
LB medium (See Appendix) containing the appropriate antibiotic. For
DAS constructs, 50 mg/l spectinomycin was added to the LB medium.
The cultures were incubated for 24-48 hrs at 28.degree. C. The
duration of the incubation time was dependent upon colony size. If
pin-point colonies developed after 24 hrs, the cultures were
incubated for an additional day.
[0288] When the colonies were of a well-formed size, liquid
cultures were prepared. Four colonies were picked with a sterile
pipette tip, then added to 50 ml of YM selective medium containing
50 mg/l spectinomycin for DAS constructs (See below). Cultures were
grown in a shaking incubator at 28.degree. C. and 100 rpm for 24
hrs, or until the culture reached an OD.sub.600 of 0.5-0.6. It
takes approximately 24 hrs to reach this OD. When the desired OD
was reached, the cells were centrifuged at 8000 rpm for 10 min at
20.degree. C. The pellet was resuspended in MS liquid medium at the
same original volume as the YM selective medium.
[0289] Infection. Cotyledon explants were cultured on the feeder
layer plates 1 day prior to infection with Agrobacterium. For
infection, they were incubated in the Agrobacterium suspension for
10 min, then the suspension was removed. The explants were blotted
on sterile paper towels, then placed with the adaxial sides down on
the original feeder plate cultures. They were maintained at
19.degree. C. in the dark for 48 hrs of cocultivation.
[0290] Plant regeneration: After cocultivation, cotyledon explants
were cultured with the adaxial sides up on selective 2Z medium
containing 3 mg/l bialaphos. The cultures were maintained at
24.+-.2.degree. C. under a 16-hr photoperiod of cool white
fluorescent lights. Three weeks later, the cultures were
transferred to 1Z medium containing 3 mg/l bialaphos (See below),
then to fresh medium at 3 week intervals. When shoots began to
regenerate, the cultures were transferred to the same 1Z medium
with bialaphos in Magenta boxes. When shoots were 2 cm tall, they
were transferred to selective rooting medium containing 2 mg/l
bialaphos (See below) in Magenta boxes. Plants were maintained at
24.+-.1.degree. C. under a 16-hr photoperiod of cool white
fluorescent lights. After approximately 3 weeks, cuttings from
these plants were transferred again to selective rooting medium
containing bialaphos, however, timentin was not included to
determine if there was Agrobacterium contamination present.
[0291] Analysis. Plants that rooted on selective rooting medium
were selected for ELISA analysis. Leaf material was harvested,
transferred to 2 ml conical screw cap tubes, and placed on ice.
ELISA was performed at least twice before selecting the lines
containing the highest antigen level. The elite lines were
propagated and transferred to the greenhouse.
[0292] Greenhouse acclimation. Plants were transferred to the
greenhouse when they had a well-developed root system. The agar
medium was washed off the roots before transferring the plants to
6-inch pots containing Cornell mix. They were covered with plastic
domes. During the next week, the domes were gradually lifted to
acclimate the plants. After approximately 5 weeks, the plants are
transferred to 3-gallon pots containing Cornell mix.
Media Ingredients
1/2 MSO
TABLE-US-00014 [0293] Per liter MS salts 2.15 g Myoinositol 100 mg
Thiamine HCl stock (0.4 mg/ml) 5 ml Pyridoxine HCl stock (0.5
mg/ml) 1 ml Nicotinic acid stock (0.5 mg/ml) 1 ml Sucrose 10 g pH
to 5.8 .+-. 0.03 Agar/Agar 8 g
KCMS
TABLE-US-00015 [0294] Per liter MS salts 4.3 g Thiamine HCl stock
(1 mg/ml) 1.3 ml Myoinositol 100 mg 2,4-D stock (1 mg/ml) 200 .mu.l
KH.sub.2PO.sub.4 200 mg Kinetin stock (1 mg/ml) 100 .mu.l Sucrose
30 g pH to 5.5 .+-. 0.03 Agargel 5.2 g
LB
TABLE-US-00016 [0295] Per liter Bacto-tryptone 10 g Yeast extract 5
g NaCl 10 g Difco Bacto Agar 15 g
YM
TABLE-US-00017 [0296] Per liter Yeast extract 400 mg Mannitol 10 g
NaCl 100 mg MgSO.sub.4.cndot.7H.sub.20 200 mg KH.sub.2PO.sub.4 500
mg
Alternatively, YM in powder form can be purchased (Gibco BRL,
catalog #10090-011). To make liquid culture medium, add 11.1 g to 1
liter water.
MS Liquid Medium
TABLE-US-00018 [0297] Per liter MS salts 4.3 g Myoinositol 100 mg
Glycine 2 mg Nicotinic acid 0.5 mg Pyridoxine HCl 0.5 mg Thiamine
HCl 0.4 mg Folic acid 0.25 mg D-biotin 0.05 mg Sucrose 30 g pH
5.6
2Z
TABLE-US-00019 [0298] Per liter MS salts 4.3 g Myoinositol 100 mg
Nitsch vitamins stock (1000X)* 1 ml Sucrose 20 g pH to 6.0 .+-. 0.3
Agargel 5.2 g
Selective Rooting Medium
TABLE-US-00020 [0299] Per liter MS salts 4.3 g Nitsch vitamins
stock (1000x)* 1 ml Sucrose 30 g pH to 6.0 + 0.03 Difco Bacto Agar
8 g
Add the following filter-sterilized components per liter after
autoclaving: Bialaphos: 2 ml of a 1 mg/ml stock solution Timentin:
3 ml of a 100 mg/ml stock solution
Nitsch Vitamins Stock (1000.times.)
TABLE-US-00021 [0300] Per 50 ml Glycine 0.1 g Nicotinic acid 0.5 g
Pyridoxine HCl 0.025 g Thiamine HCl 0.025 g Folic acid 0.025 g
d-biotin 0.002 g
[0301] Adjust pH to 7.0 to clear solution.
EXAMPLE 12
Tomato as a Production System of Edible Vaccines
[0302] Assembly of a Synthetic HN Gene. A HN expression cassette
that includes the promoter of the Casava vein mosaic virus (CsVMV)
and terminated by the 3' element of the Soybean Vegetative Storage
Protein (VSP) was assembled and inserted into binary vectors by the
Mason Laboratory (The Boyce Thompson Institute for Plant Research
(BTI)) for Agrobacterium-mediated plant transformations. The vector
carries the gene encoding the plant selection marker
phosphinothricin acetyl transferase (PAT, described in U.S. Pat.
Nos. 5,879,903; 5,637,489; 5,276,268; and 5,273,894) (FIG. 30).
[0303] Tomato Transformation and Regeneration.
Agrobacterium-mediated transformation of tomato cotyledons (variety
Tanksley TA234TM2R) was performed according to Frary and Earle [12]
by the Van Eck Laboratory (BTI). Regenerating explants were
transferred to fresh medium every 3 weeks. Green shoots were
transferred to GA-7 boxes (Magenta Corporation, Chicago, Ill.)
containing rooting media (MS salts 4.3 g, 1 ml/l Nitsch vitamins
1000.times., 30 g/l sucrose, 8 g/l difco bacto agar, pH 6.0,
supplemented with 50 mg/l kanamycin, 300 mg/l timentin and 4 mg/l
IBA) when they were approximately 1 cm tall. Rooted plantlets were
transferred to soil and maintained at 28.degree. C. under a 12
hours photoperiod.
[0304] Protein Extraction and Elisa Analysis. Crude protein
extracts were made by homogenizing 1 mg of fresh leaf, fruit or NT1
cell material per 5 .mu.L of PBS or 1 mg of dried leaf, fruit or
NT1 cell material per 10 .mu.L of PBS in a QBiogene (Carlsbad,
Calif., USA) Fast Prep machine. Insoluble material was removed by
centrifugation at 14,000 rpm in an Eppendorf 5415C microcentrifuge
at 4.degree. C. for 5 minutes. The resulting sample supernatants
were kept on ice during analysis and subsequently stored at
-80.degree. C.
[0305] Ninety-six well, microtiter plates (Costar 3590, Fisher
Scientific, PA, USA check) were coated with 100 .mu.l per well of a
1 in 1,000 dilution of SPAFAS chicken anti-NDV polyclonal antibody
(Benchmark Biolabs, N E) in 0.01 M borate buffer. The plates were
covered and incubated overnight at 4.degree. C. The plates were
equilibrated to room temperature for 30 minutes then washed three
times with 300 .mu.l per well phosphate buffered saline with 0.05%
Tween-20 (PBST). The plates were blocked with 200 .mu.l per well,
3% skim milk in PBST at 37.degree. C. for two hours then washed
three times with PBST before 50 .mu.l per well of protein extracts
were added. ELISAs were performed on two replicates on a series of
two-fold dilutions of the crude extracts in 5% skim milk+PBS+0.05%
Tween-20. The plates were incubated for one hour at 37.degree. C.
before washing three times with PBST. One hundred microliters of
the primary antibody, HN Mab 4A (Benchmark Biolabs), diluted 1 in
250 in 1% skim milk in PBST, was added to each well and incubated
for one hour at 37.degree. C. The plates were then washed three
times with PBST before 100 .mu.l per well of goat, anti-mouse IgG
horse radish peroxidase (HRP) conjugate (Sigma, St Louis, Mo., USA)
diluted 1 in 3,000 in 1% skim milk in PBST was added and left to
incubate at 37.degree. C. for one hour. The plates were washed four
times with PBST before 50 .mu.l per well of TMB Peroxidase EIA
Substrate kit (BioRad) was added and incubated for five minutes at
room temperature. Absorbance at 450 nm was measured in a ThermoMax
Micropla reader. ELISA data obtained by anti-HN ELISA was converted
to microgram per gram of fresh weight by reference to a standard
curve constructed using purified HN (Benchmark Biolabs).
[0306] The top four lines based on HN expression in the leaves were
used for fruit analysis.
[0307] Nucleic Acid Extraction. Ten milliliters of extraction
buffer (4% p-amino salicylic acid, 1% 1,5 naphthalenedisulfonic
acid, disodium salt hydrate), 3 ml CTAB buffer, and 10 ml
buffer-saturated phenol (pH 4.3) were added to a 50 ml falcon tube
and heated at 70.degree. C. in a water bath for 10 minutes. About
3.5 g of individual tomato fruit were ground in liquid nitrogen
then added to the heated tube and vortexed vigorously for 30
seconds. Ten milliliters of chloroform:isoarnylalcohol (24:1) was
added. The resulting slurry was vortexed for 30 seconds before
centrifuging for 20 minutes at 10,000 rpm at 4.degree. C. The
aqueous phase was transferred to a 50 mL falcon tube, mixed with 2
volumes of ethanol, and precipitated for 15 minutes at room
temperature. The extract was then centrifuged for 15 minutes at
10,000 rpm at 4.degree. C. before the supernatant was discarded.
The resulting nucleic acid pellet was resuspended in 2 ml DEPC
treated water, mixed with an equal volume of 4 M LiCl, and
precipitated at -20.degree. C. overnight. The extract was
centrifuged at 10,000 rpm at 4.degree. C. for 20 minutes. The
supernatant containing genomic DNA was removed to a different tube,
precipitated with 2 volumes of ethanol and stored at -20.degree. C.
overnight. Meanwhile, the RNA pellet was resuspended in DEPC
treated water and stored at -20.degree. C. The following day, the
DNA pellet was centrifuged down at 10,000 rpm at 4.degree. C. for
20 minutes and resuspended in water containing 1 .mu.g/ml Rnase
A.
[0308] Southern Analysis. Fifteen micrograms of tomato genomic or
330 ng of pCHN plasmid DNA were digested with 5 units of
restriction enzyme EcoRI per .mu.g DNA at 37.degree. C. for 20 to
24 hours. Uncut and digested samples were run overnight in a 1.0%
TAE agarose gel. The gel was prepared for transfer by one 20 minute
depurination wash (0.25 M HCl), two 30 minute denaturation washes
(1.5 MNaCl, 0.5 MNaOH) and two 30 minute neutralization washes (0.5
M Tris HCl, pH 7, 3 MNaCl). DNA was then transferred to a nylon
membrane (Zeta-Probe blotting membranes, Bio-Rad, Hercules, Calif.,
USA) by capillary transfer and fixed by UV cross-linkage using a
Bio-Rad GS Gene Linker. A PCR labeled probe was made by using the
primer set HNa (CCG AGC AGT TTC ACA AGT GG, SEQ ID NO: 10) and HNb
(CCT GAT CTT GCT TCA CGT ACA, SEQ ID NO: 11) on a pCHN template.
DIG labeled dCTP was incorporated into the 1734 bp amplicon using
the Roche Molecular Biochemical DIG PCR Probe Synthesis kit
according to manufacturer's instructions. The amplification was
performed over 37 cycles using an iCycler Gradient Thermo Cycler
(BioRad, Hercules, Calif., USA). The template was initially melted
at 94.degree. C. for 5 minutes followed by 35 cycles of 94.degree.
C. for 30 seconds, 56.degree. C. for 30 seconds, and 72.degree. C.
for 90 seconds. A final extension step was performed at 72.degree.
C. for 5 minutes before soaking at 4.degree. C. Hybridization
bottles and 10 ml DIG Easy Hyb (Roche Scientific, Mannheim,
Germany) per membrane were pre-warmed to 45.degree. C. in a
hybridization oven. Membranes were prehybridized for 60 minutes at
45.degree. C. and hybridized overnight at 45.degree. C. with a
probe concentration of 5 .mu.l/ml DIG Easy Hyb.
[0309] Post hybridization washes and detection were performed as
per manufacturer's instructions (Roche--DIG wash and block buffer
set and DIG Luminescent Detection Kit). Labeled membranes were
visualized after exposure to film.
[0310] Northern Analysis. Thirty micrograms of total RNA from
tomato transformants and wild type plants and 1.25 .mu.g of ladder
(high range RNA ladder, MBI Fermentas, Hanover, Md.) were denatured
with formaldehyde/formamide and run for two hours in a 1% agarose
3-(N-morpholino) propanesulfonic acid (MOPS) formaldehyde gel at
80V. The RNA was transferred to zeta probe membrane (BioRad,
Hercules, Calif., USA) by upward capillary action and fixed by UV
cross-linkage. The membrane was then stained with 0.04% methylene
blue in 0.5M sodium acetate to determine if RNA transfer was
successful and to confirm that RNA concentrations in all samples
were similar. A PCR labeled, DNA probe was made using the primer
set HNa and HNb on a pCHN template. DIG labeled dCTP was
incorporated into the 1734 bp amplicon as per manufacturer's
instructions (PCR DIG Probe Synthesis kit, Roche Scientific,
Mannheim, Germany). The amplifications were performed as described
for Southern analysis. Hybridization bottles and 10 ml DIG Easy Hyb
(Roche) per membrane were pre-warmed to 45.degree. C. in a
hybridization oven. Membranes were pre-hybridized for at least 90
minutes at 45.degree. C. and hybridized overnight with a probe
concentration of 7.5 .mu.l/ml DIG Easy Hyb. Post hybridization
washes and detection were performed as per manufacturer's
instructions (Roche--DIG wash and block buffer set and DIG
Luminescent Detection Kit). Labeled membranes were visualized after
exposure to film.
[0311] Western Analysis. Purified HN supplied by Benchmark Biolabs
and NT1 cell line 119 transformed with pCHN (supplied by BTI) were
used as positive controls. Twenty microliters of protein extracts
were added to 4 .mu.l 6.times.SDS gel loading buffer (300 mM
Tris-HCl, pH 6.8, 600 mMDTT, 12% SDS, 0.6% bromophenol blue, 60%
glycerol), boiled for 10 minutes and loaded into a 15% sodium
dodecyl sulfate polyacrylamide gel. The gel was run in tris-glycine
buffer (25 mM Tris, 250 mM Glycine, pH 8.3, 0.1% SDS) at 30
milliamps per gel until the dye front ran about 5 mm from the gel
bottom. The separated proteins were transferred from the gel to a
PVDF membrane using a BioRad Trans Blot Cell (50 V for 2 hours or
overnight at 7 V). All membrane washes were performed in PBST
(PBS+0.1% Tween-20) at room temperature unless otherwise stated.
The membrane was blocked with 5% skim milk+PBS+0.1% Tween-20
overnight at 4.degree. C. or for two hours at room temperature
using slow rotation in a hybridization incubator (Fisher
Scientific, Tustin, Calif., USA). The membrane was washed twice
briefly before incubating for one hour at 37.degree. C. with a 1 in
50,000 dilution of the primary antibody, mouse anti-HN Mab14F
antiserum (Benchmark Biolabs) in 1% skim milk+PBS+0.1% Tween-20.
The membrane was briefly rinsed in PBST before a 15 minute wash and
three 5 minute washes then incubated in a 1 in 30,000 dilution of
an anti-rabbit IgG horseradish peroxidase (HRP) conjugate (Sigma)
for an hour at 37.degree. C. with slow rotation. The membrane was
rinsed, then subjected to a 15 minute wash and three 5 minute
washes. Detection was performed using the Amersham ECL+kit as per
manufacturer's instructions.
[0312] Haemagglutination Activity. To make a 1% chicken red blood
cell (cRBC) standardized solution, cRBCs in Alsevers solution
(Colorado Serum, Co) were transferred into a 15 ml conical tube and
centrifuged at 250 g for 10 minutes. The supernatant was aspirated
and the pellet resuspended in 10 ml Dulbecco's phosphate-buffered
saline without calcium and magnesium (DPBS) (Cellgro, Mediatech,
Inc, Kansas City, Mo.). The suspension was centrifuged at 250 g for
10 minutes. The washes and resuspensions were repeated until the
supernatant was clear. Once this was achieved the cells were
pelleted and the supernatant aspirated to leave the packed RBC
pellet. The pellet was then diluted in 1% DPBS-(volume to volume).
Four hundred microliters of the 1% RBC solution was transferred to
a small tube, 1.6 ml of deionized water was added before being
vortexed at high speed for 20 seconds to lyse the cells. A cRBC
solution was not used unless the absorbance at 540 nm of the lysed
cells was 0.4-0.5. A 96-well, U bottom plate (Falcon) was sprayed
with antistatic spray before 50 .mu.l per well of DPBS was added.
Fifty microliters of the samples, including the positive control of
NDV HN and negative control of DPBS, was added to the first row,
mixed through repeated pipetting then serially diluted by
transferring 50 .mu.l to the next row. Fifty microliters of the
standardized cRBC was added to each well before the dilutions were
incubated on a plate shaker at 600 rpm for 20-30 seconds. The
plates were then incubated at 5.degree. C. for an hour before the
control NDV HN wells were checked for haemagglutination. Once this
was achieved the final read was made. The HA titer was taken as the
reciprocal of the highest dilution that was positive for
agglutination.
[0313] Analysis. Fruit ripening is a developmentally and
genetically regulated process that is characterized by many
biochemical and physiological changes, including increases in the
rate of ethylene biosynthesis and respiration, chlorophyll
degradation, pigment accumulation, textural modifications such as
fruit softening, changes in the levels of sugars and organic acids,
and production of volatile aromatic compounds (Brady CJ. Annu Rev.
Plant Physiol. 1987; 38: 155-178). There is a distinct relationship
between fruit pH and solids content (mainly sugars, Benton Jones J.
Tomato plant culture: in the field, greenhouse, and home garden.
New York: CRC Press, 1999). The degree of ripeness is also a factor
that affects pH. Ripening of wild type TA234 caused the fruit pH to
decrease significantly (.alpha.=0.05) (FIG. 31) and the total
soluble protein to generally decrease (FIG. 32). Although the
extent of decrease varies, this has been found in other studies
(Benton Jones J. Tomato plant culture: in the field, greenhouse,
and home garden. New York: CRC Press, 1999).
[0314] The four lines expressing the highest level of HN in leaf
tissue had varying phenotypes. The phenotypes of lines CHN-1,
CHN-12 and CHN-32 were indistinguishable from control plants while
line CHN-10 showed traits indicative of polyploidy such as thick,
wrinkly leaves and late flower set and fruit development. Comparing
the intensity of signals between the plasmid controls and the
genomic samples in Southern analysis, the lines had between 1 and 4
copies of the transgene (FIG. 33) with CHN-1 having two copies,
CHN-10 four copies, CHN-12 having one copy, and CHN-32 having 2
copies. Since CHN-10 is likely a polyploid line, it will not be
used in further studies or for production of vaccine batches.
[0315] Methylene blue staining of the northern membrane before the
pre-hybridization and hybridization steps revealed the transfer of
RNA from the gel to the membrane was successful and that there were
similar concentrations of RNA in each of the samples (FIG. 34a).
Northern analysis of total RNA specific for the HN gene
demonstrated no band in the wild type negative control however a
band of about 5000 nucleotides was seen in the transgenic tomato
lines (FIG. 34b). Since the HN gene expression cassette with
promoter and terminator is 2,832 bp and the expression cassettes
within the T-DNA combined are 4323 bp, the transcript is larger
than expected and was thought due to read through of the VSP 3'
termination signal during transcription of the HN gene. Transcript
prevalence varied at different stages of fruit ripening within a
line and between lines. Line CHN-10 displayed a distinct decrease
in HN-specific mRNA content as the fruit ripened (FIG. 34b).
However since only one fruit was sampled for northern analysis no
pattern could be discerned between RNA prevalence and stage of
ripening in the other CHN tomato lines.
[0316] Anti-HN ELISA of transgenic lines revealed a trend of
decreasing concentration as the fruit ripened (FIG. 35). Stages 4
and 5 had significantly less HN per gram fresh weight than stage 1
fruit in lines CHN-1 (FIG. 35a) and CHN-10 (FIG. 35b) and stage 2
fruit in CHN-32 (FIG. 35c) (.alpha.=0.05). HN concentration was not
significantly different at the same stage between different
varieties (.alpha.=0.05). HN concentration varied between 71.1 and
3.5 .mu.g/g fresh weight with the highest concentration in each
line being 67.2 .mu.g/g fresh weight in stage 1 fruit of CHN-1,
63.3 .mu.g/g fresh weight in stage 1 fruit of CHN-10, 65.4 .mu.g/g
fresh weight in stage 1 fruit of CHN-12 and 71.1 .mu.g/g fresh
weight in stage 2 fruit of CHN-32. The best stage to harvest a HN
tomato crop would therefore be at the green or early breaker
stage.
[0317] Western analysis of the Lasota NDV HN positive control while
overloaded, revealed bands around 49, 74, 78, 90, 120, 200, 250 and
larger than 250 kDa (FIG. 36). The 78 kDa band was designated HN
monomers, the smaller bands degradation products. The 90 kDa band
was thought a different glycosylation form and the 120 kDa and
larger bands as HN polymers. Western analysis of transgenic fruit
at different stages was difficult due to the low expression of HN,
however two bands around 78 and 70 kDa were visible. These bands
were also present in the leaves of transgenic tomato plants and in
the NT1 cell line 119 but were not present in the tomato or NT1
cell line negative controls. It was thought the 78 and 70 kDa bands
represented different glycosylation forms of the HN antigen or the
70 kDa band a truncated version of the HN monomer. Additional bands
thought to be degradation products were visible around 20 and 48
kDa in leaf samples of transgenic tomatoes and 48 kDa in the NT1119
line.
[0318] NF, represents tomato fruit negative control--wild type
fruit; NL tomato leaf negative control--wild type leaf, NNT NT1
cell negative control--non-transformed cell lines; 119, transgenic
NT1 cell line 119; L10, leaf from transgenic tomato line 10; L32,
leaf from tomato line 32; HN, animal derived Lasota NDV virus; M,
Bio-Rad's precision plus protein all blue standard; 1-1, fruit from
line CHN-1, stage 1 of ripening; 1-3, fruit from line CHN-1, stage
3 of ripening; 1-6, fruit from line CHN-1, stage 6 of ripening;
32-1, fruit from line CHN-32, stage 1 of ripening; 32-3, fruit from
line CHN-32, stage 3 of ripening; 32-6, fruit from line CHN-32,
stage 6 of ripening; 10-1, fruit from CHN-10, stage 1 of ripening.
Protein size is give in kDa.
[0319] Haemagglutination assays of the freeze-dried green fruit and
leaves of the transgenic tomato revealed haemagglutination activity
in all lines (FIG. 37). Activity was highest in the leaves in lines
CHN-1, CHN-12 and CHN-32 with CHN-10 being the only line to have
higher activity in the fruit. Line CHN-32 displayed the highest
haemagglutination activities of 512 and 2,048 in the fruit and
leaves (FIG. 37a) as well as the highest haemagglutination activity
of 10,928 units per microgram HN (FIG. 37b). The CHN-1 line had the
second highest haemagglutination activities of 128 and 256 in the
fruit and leaves as well as the second highest activity of 3,994
units per .mu.g HN (FIGS. 37a and b). Despite being mis-processed
during transcription, the synthetic HN gene was translated into a
functional protein.
[0320] Although CHN-10 had higher HN activity in the fruit, the
target organ for animal trials and vaccine delivery, the probable
polyploid status of this line in addition to its slowness to flower
and fruit made it an unlikely candidate for future studies. Taking
into account HN expression levels and HN activity in the four
lines, the CHN-1 and CHN-32 lines were chosen for future
analysis.
[0321] Western analysis, ELISA and haemagglutinin activity assays
show that tomato is capable of expressing a HN protein of the
correct size (78 kDa) that is antigenic in ELISAs and retains
haemagglutination activity. The optimal time to harvest tomato
fruit expressing HN under the control of the CsVMV promoter was the
early stage of fruit ripening. This decreased the time to harvest
by 2 weeks and increased HN expression approximately 15-fold.
Despite the protein being correctly processed, northern analysis
reveals that the gene is not processed correctly at the DNA level.
The 5,000 nucleotide transcript is likely due to read through of
the HN gene terminator. Lines CHN-1 and CHN-32 were deemed the best
lines for progression to additional studies.
EXAMPLE 13
HN Expression During Maturation of Tomato Fruit
[0322] HN levels in maturing tomato fruit to determine if immature
fruit are capable of expressing higher levels of HN than stage one
tomatoes.
[0323] The fruit of red-fleshy tomato varieties is said to be
mature when it has completed growth but is still completely green.
This stage of ripeness is known as "green" or "stage one". Tomatoes
are usually picked at this stage then ripened with ethylene. The
stages following include "breakers" or "stage two" when there is a
definite break in color from green to tannish-yellow or pink (vine
ripened tomatoes are picked at this stage); "turning" or "stage
three" when more than 10% but less than 30% (for example, 11, 12,
13, 14, 15, 20, 25, 29%) of the surface of the tomato shows a
definite change in color from green to tannish-yellow, pink or red;
"pink" or "stage four" when more than 30% but less than 60% (for
example, 31, 32, 33, 34, 35, 40, 45, 50, 55, 59%) of the surface of
the fruit is pink or red in color; "light red" or "stage five" when
more than 60% but less than 90% (for example, 61, 62, 63, 64, 65,
70. 75, 80, 85, 89%) of the surface of the fruit is pinkish-red or
red; "red" or "stage six" when more than 90% (for example, 91, 92,
93, 94, 95, 99, 100%) of the surface of the fruit is red.
[0324] Expression of the synthetic, plant optimized, gene for the
Newcastle disease virus haemagglutinin neuraminidase (H) protein
driven by the Casava Vein Mosaic Virus promoter (CsVMV), decreased
as the tomato fruit ripened. It was determined that in stage one
green tomato fruit, HN expression was approximately 12 .mu.g/g
fresh weight (FW) and that this steadily decreased as the tomato
ripened to stage six red tomato fruit to an approximate value of
2.5 .mu.g/g FW.
[0325] One T.sub.1 plant from each of the elite lines in the
T.sub.0 CHN generation (CHN-1, CHN-32) was germinated and allowed
to grow. When flowering began, cross-pollination was prevented by
each flower being self-pollinated by hand and enclosed in a paper
towel. The individual flower was dated and allowed to fruit. Three
fruit from each plant line were harvested at one week
post-pollination, two weeks post-pollination, four weeks
post-pollination, six weeks post-pollination, and finally at stage
one green fruit (about eight weeks post pollination). Fruit
diameter (mm) and fresh mass (g) were recorded before ELISA
analysis of the HN content and lyophilization. To measure the
diameter of the tomato fruit, a vernier caliper was applied to the
widest part of the tomato fruit perpendicular to the stem and the
measurement in millimeters recorded. Mass was determined using a
gram balance.
[0326] HNELISA. SPAFAS chicken A-NDV polyclonal antibody diluted
1:1500 in 0.01 M borate buffer was used to coat a 96 well ELISA
plate. One hundred microliters of the dilution was pipetted into
each well before the plate was covered, and left overnight at
4.degree. C. The next morning the plate was allowed to equilibrate
for 30 minutes at 24.degree. C., before being washed three times
with PBST (0.05% tween). A solution of 3% skim milk was made and
200 .mu.l added to each well. The plate was placed at 37.degree. C.
and allowed to block for two hours.
[0327] To collect a sample, a coring tool (size 1) was pushed
through the center of the tomato along the horizontal axis. Any
gelatinous material or seeds were excluded from the sample. Using a
scalpel, approximately 1 cm of the tomato was collected and placed
into the sample tube that was then placed on ice. The actual sample
weight was calculated by subtracting the individual tube weight
from the total mass then 20.times. tomato extraction buffer (4M
Nacl (final concentration 100 mM), 0.5M EDTA (final
concentration1mM), 20% Triton-x 100 (final concentration 10%),
Leupeptin (final concentration 10 ug/ml), 0.5M NaPi, pH 7.0 (final
concentration 50 mM), brought to volume with milliQ water) (mass by
volume) was added to the tube. A ceramic bead was added to the
sample tube and the sample homogenized using a fast prep machine at
speed 4.0 for 30 seconds. The homogenized samples were centrifuged
and then set aside on ice while the standard curve was
prepared.
[0328] After the plate had blocked for two hours it was washed
three times with PBST (PBS plus 0.05% tween). For the NDV HN
standard curve, 100 .mu.l of 232 ng/ml NDV purified stock (1:80)
was pipetted into the second well of the second row. Fifty
microliters of 1% skim milk was pipetted into the remaining wells
of the second row and each of the wells in rows 3-8 of the
microtiter plate. Fifty microliters of each plant sample was then
added to the 1% skim milk in wells 3-11 of the second row. The
purified NDV as well as the plant samples were then serially
diluted down the plate by pipetting 50 .mu.l out of row two and
into row three and so on down the plate. The samples were mixed in
the wells by pipetting up and down after each dilution step. The
initial concentration of the samples was a 40-fold dilution. The
plate was placed back into the 37.degree. C. incubator for 1
hour.
[0329] After incubating for one hour the plate was washed three
times with PBST and the primary antibody added. The primary
antibody NDV HN Mab 4A was diluted to a concentration of 1:250 in
1% skim milk and 100 .mu.l added to each well. This was allowed to
incubate for one hour at 37.degree. C. Next the plate was washed
three times with PBST and the secondary antibody goat anti-mouse
IgG, was added to each well at a concentration of 1:3000 in 1% skim
milk. This was allowed to incubate for one hour at 37.degree.
C.
[0330] The plate was washed four times with PBST and 50 .mu.l TMB
substrate was added to each well. After five minutes had elapsed
the TMB was neutralized with 1N H2SO.sub.4. The plate was then read
on a spectrophotometer at a wavelength of 450 nm.
[0331] The percent water loss was determine by removing the seeds
from each tomato fruit, measuring the mass, freezing at -20.degree.
C., lyophilizing, then reweighing the tomato fruit.
[0332] To take into account the increase in fruit size in addition
to HN concentration, the HN content of a tomato fruit at each of
the maturation stages selected was calculated by multiplying the
fruit HN concentration by the fruit mass. In addition, the data
generated from this study was used to calculate the possible number
of doses produced from the CHN elite lines if fruit were harvested
at stage one or at four weeks post pollination. In this model it
was assumed that the same number of fruit would be produced from a
plant harvested when fruit were at stage three and a plant
harvested when fruit were four weeks post pollination and that one
dose would be 50 .mu.g of antigen.
Results
Fruit Physiology
[0333] As expected, fruit size, mass and percent water loss
increased with time (FIGS. 38, 39, and 40). Only small variations
were observed between repetitions of measurements taken at the same
maturation stage within the same plant line. No significant
difference was seen between line 1 and 32 at the same stage in
maturation (.alpha.=0.05).
HN Content of Maturing Fruit
[0334] Concentration of HN in maturing fruit peaked at two weeks
post pollination then decreased as the fruit matured (FIG. 41).
There was no significant difference between the two tomato lines at
the same stage of maturation nor between the HN content in the
first four weeks after pollination (.alpha.=0.05). There was
significantly more HN in fruit two weeks post pollination than in
fruit six weeks after pollination and when mature and in the stage
1 of ripening (.alpha.=0.05).
[0335] To take into account the increase in fruit size in addition
to HN content, the amount of HN of a tomato fruit at each of the
maturation stages selected was calculated. The HN content within a
fruit peaked at four weeks post pollination (723 .mu.g for line
CHN-1 and 630.9 .mu.g for line CHN-32) before decreasing with
further fruit maturation (FIG. 42). There was no significant
difference between lines at the same stage of maturation
(.alpha.=0.05).
[0336] Calculations were made for the number of doses produced by
the HN elite lines if fruit were harvested at stage 1 or four weeks
post pollination (Table 10). It was assumed the same number of
fruit would be produced from each harvest and that one dose would
be 50 .mu.g of antigen. These data indicate that if 33 fruit are
harvested at stage one, 126 doses would be produced; and if fruit
were harvested four weeks post pollination, 486 doses would be
produced. Thus harvest time is reduced by four weeks and there is a
286% increase in doses yielded.
TABLE-US-00022 TABLE 10 Effect of harvest time on number of HN
doses produced. Four Weeks Post Characteristic Stage 1 Pollination
Antigen Concentration 2.5 18.4 (.mu.g/g) Average Weight 76.2 40
(g/fruit) Number of Fruit Harvested 33 33 Total Mass of Fruit
2514.6 1320 Produced (g) Total Mass of Antigen 6.3 24.3 (mg) Number
of 50 .mu.g Doses 126 486
[0337] Although HN concentration peaked at 38.8-42 .mu.g/g fresh
weight in tomato fruit two weeks post pollination, no significant
difference was found in HN concentration in the first four weeks
after pollination (.alpha.=0.05). When mass was taken into account
however, tomato fruit that were four weeks post pollination
averaged between 631-723 .mu.g HN, which was a significantly higher
amount of HN than the other maturation stages tested (a=0.05).
Since percentage of water loss between fruit at stage one of
ripening and two and four weeks post pollination did not varying
greatly (total difference of 2.6%) the significantly higher antigen
content at four weeks pollination was not a factor of varying water
content or dilution of antigen. These data suggest that the best
time to harvest tomato fruit expressing HN under the control of the
CsVMV promoter is four weeks post pollination.
[0338] To identify easily when fruit are four weeks post
pollination, fruit mass and diameter were recorded throughout their
maturation. Fruit four weeks post pollination averaged a mass of 40
g and a diameter between 42 and 45 mm. Fruit size is affected by
genetics, temperature, day length and plant age. The small standard
errors of our means indicated that genetics is not presently an
issue with regards to plant-by-plant variation in fruit size.
However the effect of stress (due to change in temperature, day
length and plant age) means that fruit size may not always prove an
accurate indication of time post pollination. Large temperature
fluctuations, day length and plant age should therefore be kept in
mind, when approximating time of pollination using fruit size.
[0339] To calculate the benefit of harvesting earlier in the
maturation of tomato fruit we constructed a conservative model that
assumed 33 fruit are harvested from one plant over an average
production period, and one dose of HN would be 50%g. The model was
deemed conservative since one dose is likely to be less than 50
.mu.g and plants that have fruit harvested four weeks post
pollination would have reduced metabolic burden and would likely
produce more fruit than plants that have fruit harvested at a later
stage. These data suggest that by harvesting the fruit at four
weeks post pollination, the time required for fruit to be ready for
harvest is reduced by four weeks and there is a 286% increase in
doses yielded.
[0340] Tomato therefore is capable of expressing large quantities
of HN when harvested at an optimal time in fruit maturation.
EXAMPLE 14
Preparation of CHN-18 Master Seed
[0341] Master Seed Passage: Master Seed passage 2 was used for DNA
extraction. DNA Extraction and PCR Amplification: DNA extraction
was performed as described herein. PCR amplification for the HN and
PAT gene expression cassettes were conducted separately. There was
a 24 bp overlap between the PCR products of HN and PAT expression
cassettes.
[0342] For HN gene cassette amplification, 50 .mu.L PCR reaction
contained 2.5 units of Takara Ex Tag DNA polymerase (Takara Shuzo
Co, Shiga, Japan, catalog #RR001A), 0.2 .mu.M of each primers
(CHN01/CHN03), 5 .mu.L of 10.times. reaction buffer containing
MgCl.sub.2, 0.2 mM of each dNTP, and 200 ng of genomic DNA. The PCR
was performed with a Gen Amp PCR 9700 system, manufactured by
Applied Biosystem (Foster City, Calif.) at the following condition:
94.degree. C. for 5 min for 1 cycle, 94.degree. C. for 30 sec,
60.degree. C. 30 sec and 72.degree. C. for 3 min 30 sec for 40
cycles, 72.degree. C. for 7 min.
[0343] For PAT gene cassette amplification, a 50 .mu.L PCR reaction
contained 2.5 units of Takara Ex Tag DNA polymerase (Takara Shuzo
Co, Shiga, Japan, catalog #RR001A), 0.2 .mu.M of each primers
(CHN02/CHN04), 5 .mu.L of 10.times. reaction buffer containing
MgCl.sub.2, 0.2 mM of each dNTP, and 200 ng of genomic DNA were
used. The PCR was performed with a Gen Amp PCR 9700 system,
manufactured by Applied Biosystem (Foster City, Calif.) at the
following condition: 94.degree. C. for 5 min for 1 cycle,
94.degree. C. for 30 sec, 56.degree. C. 30 sec and 72.degree. C.
for 2 min 30 sec fro 40 cycles, 72.degree. C. for 7 min.
Cloning of PCR Products: After agarose gel electrophoresis and
visual observation, PCR products were purified using MiniElute PCR
Purification Kit (Qiagen, Valencia, Calif. Catalog #28004)
according to the manufacturer's protocol. Purified PCR products
were cloned into pCR.RTM. II-TOPO vector using TOPO TA Cloning.RTM.
kit (Invitrogen, Carlsbad, Calif., Catalog #051302) according to
the manufacturer's protocol. Plasmid DNA Extraction Plasmid DNA was
extracted using Qiaprep Spin Minprep kit (Qiagen, Valencia, Calif.,
catalog #27106) according to the manufacturer's protocol. DNA
Sequencing and Analysis: Plasmid DNAs containing cloned PCR
products were sent to Lark Technologies Inc (Houston, Tex.) for
sequencing using ABI PRISM.RTM. BigDye.TM. Primer Cycle Sequencing
Kits (Applied Biosystem, Foster City, Calif.). DNA sequences were
analyzed using Vector NT1 program (InforMax, Frederick, Md.).
Results
[0344] DNA sequences from the HN and PAT cassettes were assembled
and compared with the sequences from a virtual plasmid map of pCHN.
DNA sequences of all the genetic elements including the CsVMV
promoter, HN and PAT coding sequences, vspB 3' UTR, and MAS 3' UTR
were identical to the ones in the virtual plasmid map pCHN. Based
on the virtual plasmid map pCHN, the PCR product including the
whole gene insert in CHN-18 Master Seed using primer CHN01/CHN02
was 4757 bp. However, the actual cloned and sequenced PCR product
including the whole gene insert in CHN-18 Master Seed was 4768 bp.
By sequence comparison, 7 additional DNA bases were located in the
junction region (poly cloning site) between the PAT coding
sequences and MAS 3' UTR, another additional 4 DNA bases were
located outside of the 3' end of MAS3' UTR (FIG. 43). The
inconsistenct DNA base number between the virtual plasmid map and
the actual sequencing data most likely occurred during the virtual
creation of plasmid map pCHN. Open reading frame analysis of the
entire insert sequence indicated there were only the expected HN
and PAT open reading frames, and the 11 DNA bases did not result in
any changes in the existing HN and PAT open reading frame and did
not create any new opening reading frames.
CONCLUSIONS
[0345] By comparison with the virtual sequence from plasmid map
pCHN, the actual DNA sequence of the whole gene insert in CHN-18
Master Seed was identical to what was expected except for extra 11
DNA bases outside all the genetic elements in the gene insert.
These 11 DNA bases do not have any effect on the existing HN and
PAT open reading frames.
[0346] The principles, preferred embodiments and modes of operation
of the present invention have been described in the foregoing
specification and examples. The invention that is intended to be
protected herein, however, is not to be construed as limited to the
particular forms specifically disclosed, since they are to be
regarded as illustrative rather than restrictive. Variations and
changes may be made by those skilled in the art without departing
from the spirit and scope of the invention.
[0347] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of molecular biology,
cell biology, microbiology and recombinant DNA techniques, which
are within the skill of the art. Such techniques are explained
fully in the literature. See, e.g., Sambrook, Fritsch &
Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Second
Edition; Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Nucleic
Acid Hybridization (B. D. Harnes & S. J. Higgins, eds., 1984);
A Practical Guide to Molecular Cloning (B. Perbal, 1984); (Harlow,
E. and Lane, D.) Using Antibodies: A Laboratory Manual (1999) Cold
Spring Harbor Laboratory Press; and a series, Methods in Enzymology
(Academic Press, Inc.); Short Protocols In Molecular Biology,
(Ausubel et al., ed., 1995).
[0348] All patents, patent applications, and published references
cited herein are hereby incorporated by reference in their
entirety. While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
scope of the invention encompassed by the appended claims.
Sequence CWU 1
1
2611760DNANewcastle disease virus 1ccatggaccg agcagtttca caagtggctc
ttgagaatga tgagagggaa gccaagaaca 60cttggaggct tatctttcgg atagccattc
tctttcttac tgttgtcacc ctagcaatct 120ctgttgcatc attactctat
tctatgggag caagcacccc ctcagactta gttggcatac 180ccacacgaat
ctctagggct gaagagaaga ttaccagtac cctaggctcc aaccaggatg
240ttgtggaccg aatctacaaa caagttgcac ttgaaagtca acttgcatta
ctcaacacag 300aaactaccat catgaatgca atcaccagcc tatcctatca
gatcaatggg gctgccaaca 360attcaggttg gggagcccca attcatgatc
cagactacat tggaggtatt ggcaaagaac 420tcattgtaga tgatgcttca
gatgttacat ctttctatcc ttcagctttc caggaacatc 480tgaacttcat
tcctgcaccc acaactggga gtgggtgcac tcggataccc tcatttgaca
540tgagtgctac acactattgc tatacacaca atgtcattct atctggctgt
cgtgaccatt 600ctcactctta tcagtactta gcacttggag ttcttcgtac
atctgctact ggtagagtgt 660tcttctcaac tcttcgcagt atcaatcttg
atgatacaca gaatcgcaaa agttgctctg 720tatctgctac acctttgggc
tgtgatatgc tatgcagtaa agtaacagaa actgaagaag 780aggactacaa
ttctgcagtc cctacaagga tggtgcatgg cagattgggt tttgatggtc
840aataccatga gaaagatttg gatgtcacta cattgtttgg ggattgggta
gctaactatc 900caggagttgg aggtggtagc ttcattgact ccagagtctg
gttctctgtc tatggtggtt 960tgaaacctaa cagtcctagt gatactgtgc
aagagggaaa gtatgttatc tacaagaggt 1020acaatgatac ttgtcctgat
gagcaagact atcagattcg aatggctaag tcatcataca 1080aaccaggaag
atttggaggt aagaggatac aacaagctat tctcagtatc aaggttagca
1140catcattggg agaagatcca gtccttactg ttccaccaaa cactgtaaca
ttgatgggag 1200ctgagggaag gattcttact gttggtacta gccactttct
ctatcaacgt ggaagttcct 1260actttagccc agcgttactg tatccaatga
ctgtgagcaa caagacagct acattacatt 1320caccatatac tttcaatgcc
tttacaagac ctggatcgat tccttgccaa gcttcagcta 1380gatgtccgaa
ttcgtgtgtg actggagttt acactgatcc ttaccctttg atcttctacc
1440gtaatcatac cttgagaggg gtgtttggaa caatgttaga tggtgttcaa
gctaggttga 1500atcctgcctc tgctgtgttt gattctacat ccagatcaag
gataaccaga gtttcctcta 1560gttctactaa ggcagcatac actacctcca
catgtttcaa agttgtaaag acgaacaaga 1620cctattgtct gagcatagct
gagatttcta acactctctt tggggaattc agaattgttc 1680cacttttggt
ggagattctg aaagatgatg gtgtacgtga agcaagatca ggttaagtct
1740tcggatccgg taccgagctc 17602577PRTNewcastle disease virus 2Met
Asp Arg Ala Val Ser Gln Val Ala Leu Glu Asn Asp Glu Arg Glu1 5 10
15Ala Lys Asn Thr Trp Arg Leu Ile Phe Arg Ile Ala Ile Leu Phe Leu
20 25 30Thr Val Val Thr Leu Ala Ile Ser Val Ala Ser Leu Leu Tyr Ser
Met 35 40 45Gly Ala Ser Thr Pro Ser Asp Leu Val Gly Ile Pro Thr Arg
Ile Ser 50 55 60Arg Ala Glu Glu Lys Ile Thr Ser Thr Leu Gly Ser Asn
Gln Asp Val65 70 75 80Val Asp Arg Ile Tyr Lys Gln Val Ala Leu Glu
Ser Pro Leu Ala Leu 85 90 95Leu Asn Thr Glu Thr Thr Ile Met Asn Ala
Ile Thr Ser Leu Ser Tyr 100 105 110Gln Ile Asn Gly Ala Ala Asn Asn
Ser Gly Trp Gly Ala Pro Ile His 115 120 125Asp Pro Asp Tyr Ile Gly
Gly Ile Gly Lys Glu Leu Ile Val Asp Asp 130 135 140Ala Ser Asp Val
Thr Ser Phe Tyr Pro Ser Ala Phe Gln Glu His Leu145 150 155 160Asn
Phe Ile Pro Ala Pro Thr Thr Gly Ser Gly Cys Thr Arg Ile Pro 165 170
175Ser Phe Asp Met Ser Ala Thr His Tyr Cys Tyr Thr His Asn Val Ile
180 185 190Leu Ser Gly Cys Arg Asp His Ser His Ser Tyr Gln Tyr Leu
Ala Leu 195 200 205Gly Val Leu Arg Thr Ser Ala Thr Gly Arg Val Phe
Phe Ser Thr Leu 210 215 220Arg Ser Ile Asn Leu Asp Asp Thr Gln Asn
Arg Lys Ser Cys Ser Val225 230 235 240Ser Ala Thr Pro Leu Gly Cys
Asp Met Leu Cys Ser Lys Val Thr Glu 245 250 255Thr Glu Glu Glu Asp
Tyr Asn Ser Ala Val Pro Thr Arg Met Val His 260 265 270Gly Arg Leu
Gly Phe Asp Gly Gln Tyr His Glu Lys Asp Leu Asp Val 275 280 285Thr
Thr Leu Phe Gly Asp Trp Val Ala Asn Tyr Pro Gly Val Gly Gly 290 295
300Gly Ser Phe Ile Asp Ser Arg Val Trp Phe Ser Val Tyr Gly Gly
Leu305 310 315 320Lys Pro Asn Ser Pro Ser Asp Thr Val Gln Glu Gly
Lys Tyr Val Ile 325 330 335Tyr Lys Arg Tyr Asn Asp Thr Cys Pro Asp
Glu Gln Asp Tyr Gln Ile 340 345 350Arg Met Ala Lys Ser Ser Tyr Lys
Pro Gly Arg Phe Gly Gly Lys Arg 355 360 365Ile Gln Gln Ala Ile Leu
Ser Ile Lys Val Ser Thr Ser Leu Gly Glu 370 375 380Asp Pro Val Leu
Thr Val Pro Pro Asn Thr Val Thr Leu Met Gly Ala385 390 395 400Glu
Gly Arg Ile Leu Thr Val Gly Thr Ser His Phe Leu Tyr Gln Arg 405 410
415Gly Ser Ser Tyr Phe Ser Pro Ala Leu Leu Tyr Pro Met Thr Val Ser
420 425 430Asn Lys Thr Ala Thr Leu His Ser Pro Tyr Thr Phe Asn Ala
Phe Thr 435 440 445Arg Pro Gly Ser Ile Pro Cys Gln Ala Ser Ala Arg
Cys Pro Asn Ser 450 455 460Cys Val Thr Gly Val Tyr Thr Asp Pro Tyr
Pro Leu Ile Phe Tyr Arg465 470 475 480Asn His Thr Leu Arg Gly Val
Phe Gly Thr Met Leu Asp Gly Val Gln 485 490 495Ala Arg Leu Asn Pro
Ala Ser Ala Val Phe Asp Ser Thr Ser Arg Ser 500 505 510Arg Ile Thr
Arg Val Ser Ser Ser Ser Thr Lys Ala Ala Tyr Thr Thr 515 520 525Ser
Thr Cys Phe Lys Val Val Lys Thr Asn Lys Thr Tyr Cys Leu Ser 530 535
540Ile Ala Glu Ile Ser Asn Thr Leu Phe Gly Glu Phe Arg Ile Val
Pro545 550 555 560Leu Leu Val Glu Ile Leu Lys Asp Asp Gly Val Arg
Glu Ala Arg Ser 565 570 575Gly31647DNAAvian influenza virus
3gaccaaatct gcatcggtta tcatgcaaac aattcaacaa aacaagttga cacaatcatg
60gagaagaatg tgacggtcac acatgctcaa gatatactgg aaaaagagca caacgggaaa
120ctctgcagtc tcaaaggagt gaggcccctc attctgaagg attgcagtgt
ggctggatgg 180cttcttggga acccaatgtg tgatgagttc ctaaatgtac
cggaatggtc atatattgta 240gagaaggaca atccaaccaa tggcttatgt
tatccgggag acttcaatga ttatgaagaa 300ctgaagtatt taatgagcaa
cacaaaccat tttgagaaaa ttcaaataat ccctaggaac 360tcttggtcca
atcatgatgc ctcatcagga gtgagctcag catgcccata caatggtagg
420tcttcctttt tcaggagtgt ggtgtggttg atcaagaaga gtaatgtata
cccaacaata 480aagaggacct acaataacac caatgtagag gaccttctga
tattgtgggg aatccatcac 540cctaatgatg cagcggaaca aacggaactc
tatcagaact cgaacactta tgtgtctgta 600ggaacatcaa cactaaatca
gaggtcaatt ccagaaatag ctaccaggcc caaagtgaat 660ggacaaagtg
gaagaataga atttttctgg acaatactaa ggccgaacga tgcaatcagc
720tttgaaagta atgggaactt tatagctcct gaatatgcat acaagatagt
taaaaaggga 780gattcagcaa tcatgagaag cgaactggag tatggcaact
gtgataccaa atgtcagacc 840ccagtgggtg ctataaattc cagtatgcct
tttcacaatg ttcatcccct taccattgga 900gagtgtccca aatatgtcaa
atcagataaa ctggtccttg caacaggact gaggaacgtg 960cctcagagag
aaacaagagg tctgtttgga gcaatagcag gattcataga aggggggtgg
1020caaggaatgg tagatggatg gtatggttac catcatagca acgagcaggg
aagtggatat 1080gctgcagaca aagagtccac tcagaaagca atcgacggga
tcaccaataa agtcaactca 1140atcattgaca aaatgaacac tcaattcgaa
gccgttggga aagaattcaa caacttagaa 1200aggagaatag aaaatttgaa
taagaaaatg gaagatggat ttctagatgt atggacttac 1260aatgcagaac
ttctggtgct catggaaaat gaaagaactc tggatttcca tgattcatat
1320gtcaagaacc tatacgataa ggtccgactc cagctgagag ataatgcaaa
agaattgggc 1380aatgggtgtt tggagttctc ccacaaatgt gacaatgaat
gcatggaaag tgtgagaaac 1440ggaacgtatg actatccaca atactcagaa
gaatcaaggc tgaacagaga ggaaatagat 1500ggagtcaaat tggagtcaat
gggcacctat cagatactat caatttactc aacagtggcg 1560agttccctag
cactggcaat catggtagct ggtctgtctt tttggatgtg ctccaatgga
1620tcattgcaat gcagaatttg catctag 16474548PRTAvian influenza virus
4Asp Gln Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Lys Gln Val1 5
10 15Asp Thr Ile Met Glu Lys Asn Val Thr Val Thr His Ala Gln Asp
Ile 20 25 30Leu Glu Lys Glu His Asn Gly Lys Leu Cys Ser Leu Lys Gly
Val Arg 35 40 45Pro Leu Ile Leu Lys Asp Cys Ser Val Ala Gly Trp Leu
Leu Gly Asn 50 55 60Pro Met Cys Asp Glu Phe Leu Asn Val Pro Glu Trp
Ser Tyr Ile Val65 70 75 80Glu Lys Asp Asn Pro Thr Asn Gly Leu Cys
Tyr Pro Gly Asp Phe Asn 85 90 95Asp Tyr Glu Glu Leu Lys Tyr Leu Met
Ser Asn Thr Asn His Phe Glu 100 105 110Lys Ile Gln Ile Ile Pro Arg
Asn Ser Trp Ser Asn His Asp Ala Ser 115 120 125Ser Gly Val Ser Ser
Ala Cys Pro Tyr Asn Gly Arg Ser Ser Phe Phe 130 135 140Arg Ser Val
Val Trp Leu Ile Lys Lys Ser Asn Val Tyr Pro Thr Ile145 150 155
160Lys Arg Thr Tyr Asn Asn Thr Asn Val Glu Asp Leu Leu Ile Leu Trp
165 170 175Gly Ile His His Pro Asn Asp Ala Ala Glu Gln Thr Glu Leu
Tyr Gln 180 185 190Asn Ser Asn Thr Tyr Val Ser Val Gly Thr Ser Thr
Leu Asn Gln Arg 195 200 205Ser Ile Pro Glu Ile Ala Thr Arg Pro Lys
Val Asn Gly Gln Ser Gly 210 215 220Arg Ile Glu Phe Phe Trp Thr Ile
Leu Arg Pro Asn Asp Ala Ile Ser225 230 235 240Phe Glu Ser Asn Gly
Asn Phe Ile Ala Pro Glu Tyr Ala Tyr Lys Ile 245 250 255Val Lys Lys
Gly Asp Ser Ala Ile Met Arg Ser Glu Leu Glu Tyr Gly 260 265 270Asn
Cys Asp Thr Lys Cys Gln Thr Pro Val Gly Ala Ile Asn Ser Ser 275 280
285Met Pro Phe His Asn Val His Pro Leu Thr Ile Gly Glu Cys Pro Lys
290 295 300Tyr Val Lys Ser Asp Lys Leu Val Leu Ala Thr Gly Leu Arg
Asn Val305 310 315 320Pro Gln Arg Glu Thr Arg Gly Leu Phe Gly Ala
Ile Ala Gly Phe Ile 325 330 335Glu Gly Gly Trp Gln Gly Met Val Asp
Gly Trp Tyr Gly Tyr His His 340 345 350Ser Asn Glu Gln Gly Ser Gly
Tyr Ala Ala Asp Lys Glu Ser Thr Gln 355 360 365Lys Ala Ile Asp Gly
Ile Thr Asn Lys Val Asn Ser Ile Ile Asp Lys 370 375 380Met Asn Thr
Gln Phe Glu Ala Val Gly Lys Glu Phe Asn Asn Leu Glu385 390 395
400Arg Arg Ile Glu Asn Leu Asn Lys Lys Met Glu Asp Gly Phe Leu Asp
405 410 415Val Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Met Glu Asn
Glu Arg 420 425 430Thr Leu Asp Phe His Asp Ser Tyr Val Lys Asn Leu
Tyr Asp Lys Val 435 440 445Arg Leu Gln Leu Arg Asp Asn Ala Lys Glu
Leu Gly Asn Gly Cys Leu 450 455 460Glu Phe Ser His Lys Cys Asp Asn
Glu Cys Met Glu Ser Val Arg Asn465 470 475 480Gly Thr Tyr Asp Tyr
Pro Gln Tyr Ser Glu Glu Ser Arg Leu Asn Arg 485 490 495Glu Glu Ile
Asp Gly Val Lys Leu Glu Ser Met Gly Thr Tyr Gln Ile 500 505 510Leu
Ser Ile Tyr Ser Thr Val Ala Ser Ser Leu Ala Leu Ala Ile Met 515 520
525Val Ala Gly Leu Ser Phe Trp Met Cys Ser Asn Gly Ser Leu Gln Cys
530 535 540Arg Ile Cys Ile545528DNAArtificial SequencePCR primer
CVM-Asc 5atggcgcgcc agaaggtaat tatccaag 28624DNAArtificial
SequencePCR primer CVM-Xho 6atctcgagcc atggtttgga tcca
24725DNAArtificial SequenceMutagenic primer GSS-Nco 7tgccatggtg
atgtgtggtc tacaa 25823DNAArtificial SequenceForward primer GSS-1.8F
8gatctgacaa gtcaagaaaa ttg 23923DNAArtificial SequenceMutagenic
primer GSS-Xho 9agctcgagct gtgtgagtga gtg 231020DNAArtificial
SequencePrimer HNa 10ccgagcagtt tcacaagtgg 201121DNAArtificial
SequencePrimer HNb 11cctgatcttg cttcacgtac a 21124767DNAArtificial
SequenceDNA Sequence of the whole gene insert in CHN-18 Master Seed
12gaggtctaca ggccaaattc gctcttagcc gtacaatatt actcaccgga tcggccgctt
60aattaagttt aaaccctgca ggaaagcccg ggcaaaggcg cgccagaagg taattatcca
120gatgtagcat caagaatcca atgtttacgg gaaaaactat ggaagtatta
tgtgagctca 180gcaagaagca gatcaatatg cggcacatat gcaacctatg
ttcaaaaatg aagaatgtac 240agatacaaga tcctatactg ccagaatacg
aagaagaata cgtagaaatt gaaaaagaag 300aaccaggcga agaaaagaat
cttgaagacg taagcactga cgacaacaat gaaaagaaga 360agataaggtc
ggtgattgtg aaagagacat agaggacaca tgtaaggtgg aaaatgtaag
420ggcggaaagt aaccttatca caaaggaatc ttatccccca ctacttatcc
ttttatattt 480ttccgtgtca tttttgccct tgagttttcc tatataagga
accaagttcg gcatttgtga 540aaacaagaaa aaatttggtg taagctattt
tctttgaagt actgaggata caacttcaga 600gaaatttgta agtttgtgga
tccaaaccat ggaccgagca gtttcacaag tggctcttga 660gaatgatgag
agggaagcca agaacacttg gaggcttatc tttcggatag ccattctctt
720tcttactgtt gtcaccctag caatctctgt tgcatcatta ctctattcta
tgggagcaag 780caccccctca gacttagttg gcatacccac acgaatctct
agggctgaag agaagattac 840cagtacccta ggctccaacc aggatgttgt
ggaccgaatc tacaaacaag ttgcacttga 900aagtccactt gcattactca
acacagaaac taccatcatg aatgcaatca ccagcctatc 960ctatcagatc
aatggggctg ccaacaattc aggttgggga gccccaattc atgatccaga
1020ctacattgga ggtattggca aagaactcat tgtagatgat gcttcagatg
ttacatcttt 1080ctatccttca gctttccagg aacatctgaa cttcattcct
gcacccacaa ctgggagtgg 1140gtgcactcgg ataccctcat ttgacatgag
tgctacacac tattgctata cacacaatgt 1200cattctatct ggctgtcgtg
accattctca ctcttatcag tacttagcac ttggagttct 1260tcgtacatct
gctactggta gagtgttctt ctcaactctt cgcagtatca atcttgatga
1320tacacagaat cgcaaaagtt gctctgtatc tgctacacct ttgggctgtg
atatgctatg 1380cagtaaagta acagaaactg aagaagagga ctacaattct
gcagtcccta caaggatggt 1440gcatggcaga ttgggttttg atggtcaata
ccatgagaaa gatttggatg tcactacatt 1500gtttggggat tgggtagcta
actatccagg agttggaggt ggtagcttca ttgactccag 1560agtctggttc
tctgtctatg gtggtttgaa acctaacagt cctagtgata ctgtgcaaga
1620gggaaagtat gttatctaca agaggtacaa tgatacttgt cctgatgagc
aagactatca 1680gattcgaatg gctaagtcat catacaaacc aggaagattt
ggaggtaaga ggatacaaca 1740agctattctc agtatcaagg ttagcacatc
attgggagaa gatccagtcc ttactgttcc 1800accaaacact gtaacattga
tgggagctga gggaaggatt cttactgttg gtactagcca 1860ctttctctat
caacgtggaa gttcctactt tagcccagcg ttactgtatc caatgactgt
1920gagcaacaag acagctacat tacattcacc atatactttc aatgccttta
caagacctgg 1980atcgattcct tgccaagctt cagctagatg tccgaattcg
tgtgtgactg gagtttacac 2040tgatccttac cctttgatct tctaccgtaa
tcataccttg agaggggtgt ttggaacaat 2100gttagatggt gttcaagcta
ggttgaatcc tgcctctgct gtgtttgatt ctacatccag 2160atcaaggata
accagagttt cctctagttc tactaaggca gcatacacta cctccacatg
2220tttcaaagtt gtaaagacga acaagaccta ttgtctgagc atagctgaga
tttctaacac 2280tctctttggg gaattcagaa ttgttccact tttggtggag
attctgaaag atgatggtgt 2340acgtgaagca agatcaggtt aagtcttcgg
atccggtacc gagctctctc aacaatctag 2400ctagagtttg ctcctatcta
tatgtaataa ggtatgctga tatgcactat tcaaatagga 2460gcattagcta
tgtttgttaa tgtcacttta tgttatgtgg gtaagtcacc taagacactc
2520cacgtaccta cttgttgtct cttacgcggc tttaataaat cttctgccct
tgttccatat 2580ttactaatta tccctttctt cactaaaaga aaattgttat
cattaagtat tagtctttag 2640aacatatgag gtctttaatt gggtaggttt
tacaaattaa ctaatataaa atgtcataaa 2700atccacgtgg ttaaacaaat
gcagaaaatc gacgtcgtct attggaccga cagttgctat 2760taatataatg
ggccaccata gtagactgac aaataaatta cctgacaaca tcgtttcaca
2820aaaaaacaaa cacaaaaagg gagtgcattt tccagggcat ttttgtaata
aaaaacagat 2880aaaagggagt gcaatagaaa tataggggtg tggaaatagt
gatttgagca cgtcttgaag 2940cgaattcgcg gccggccaga aggtaattat
ccaagatgta gcatcaagaa tccaatgttt 3000acgggaaaaa ctatggaagt
attatgtgag ctcagcaaga agcagatcaa tatgcggcac 3060atatgcaacc
tatgttcaaa aatgaagaat gtacagatac aagatcctat actgccagaa
3120tacgaagaag aatacgtaga aattgaaaaa gaagaaccag gcgaagaaaa
gaatcttgaa 3180gacgtaagca ctgacgacaa caatgaaaag aagaagataa
ggtcggtgat tgtgaaagag 3240acatagagga cacatgtaag gtggaaaatg
taagggcgga aagtaacctt atcacaaagg 3300aatcttatcc cccactactt
atccttttat atttttccgt gtcatttttg cccttgagtt 3360ttcctatata
aggaaccaag ttcggcattt gtgaaaacaa gaaaaaattt ggtgtaagct
3420attttctttg aagtactgag gatacaactt cagagaaatt tgtaagtttg
tggatccaaa 3480ccatggcttc tccggagagg agaccagttg agattaggcc
agctacagca gctgatatgg 3540ccgcggtttg tgatatcgtt aaccattaca
ttgagacgtc tacagtgaac tttaggacag 3600agccacaaac accacaagag
tggattgatg atctagagag gttgcaagat agataccctt 3660ggttggttgc
tgaggttgag ggtgttgtgg ctggtattgc ttacgctggg ccctggaagg
3720ctaggaacgc ttacgattgg acagttgaga gtactgttta cgtgtcacat
aggcatcaaa 3780ggttgggcct aggatccaca ttgtacacac atttgcttaa
gtctatggag gcgcaaggtt 3840ttaagtctgt
ggttgctgtt ataggccttc caaacgatcc atctgttagg ttgcatgagg
3900ctttgggata cacagcccgg ggtacattgc gcgcagctgg atacaagcat
ggtggatggc 3960atgatgttgg tttttggcaa agggattttg agttgccagc
tcctccaagg ccagctaggc 4020cagttaccca gatctgaggt accctgagct
cggtcacctg tccaacagtc tcagggttaa 4080tgtctatgta tcttaaataa
tgttgtcggt attttgtaat ctcatataga ttttcactgt 4140gcgacgcaaa
aatattaaat aaatattatt attatctacg ttttgattga gatatcatca
4200atattataat aaaaatatcc attaaacacg atttgataca aatgacagtc
aataatctga 4260tttgaatatt tattaattgt aacgaattac ataaagatcg
aatagaaaat actgcactgc 4320aaatgaaaat taacacatac taataaatgc
gtcaaatatc tttgccaaga tcaagcggag 4380tgagggcctc atatccggtc
tcagttacaa gcacggtatc cccgaagcgc gctccaccaa 4440tgccctcgac
atagatgccg ggctcgacgc tgaggacatt gcctaccttg agcatggtct
4500cagcgccggc tttaagctca atcccatccc aatctgaata tcctatcccg
cgcccagtcc 4560ggtgtaagaa cgggtctgtc catccacctc tgttgcggcc
aattctgatc tggcccccat 4620ttggacgtga atgtagacac gtcgatataa
agatttccga attagaataa tttgtttatt 4680gctttcgcct ataaatacga
cggatcgtaa tttgtcgttt tatcaaaatg tactttcatt 4740ttataataac
gctgcggaca tctacat 476713681DNAHepatitis B virus 13atggagaaca
tcacatcagg attcctagga cccctgctcg tgttacaggc ggggtttttc 60ttgttgacaa
gaatcctcac aataccgcag agtctagact cgtggtggac ttctctcaat
120tttctagggg gatcacccgt gtgtcttggc caaaattcgc agtccccaac
ctccaatcac 180tcaccaacct cctgtcctcc aatctgtcct ggttatcgct
ggatgtgtct gcggcgtttt 240atcatattcc tcttcatcct gctgctatgc
ctcatcttct tattggttct tctggattat 300caaggtatgt tgcccgtttg
tcctctaatt ccaggatcaa caacaaccag tacgggacca 360tgcaaaacct
gcacgactcc tgctcaaggg aactctatgt ttccctcatg ttgctgtaca
420aaacctacgg atgggaattg cacctgtatt cccatcccat cgtcctgggc
tttcgcaaaa 480tacctatggg agtgggcctc agtccgtttc tcttggctca
gtttactagt gccatttgtt 540cagtggttcg tagggctttc ccccactgtt
tggctttcag ctatatggat gatgtggtat 600tgggggccaa gtctgtacag
catcgtgagt ccctttatac cgctgttacc aattttcttt 660tgtctctggg
tatacattta a 68114226PRTHepatitis B virus 14Met Glu Asn Ile Thr Ser
Gly Phe Leu Gly Pro Leu Leu Val Leu Gln1 5 10 15Ala Gly Phe Phe Leu
Leu Thr Arg Ile Leu Thr Ile Pro Gln Ser Leu 20 25 30Asp Ser Trp Trp
Thr Ser Leu Asn Phe Leu Gly Gly Ser Pro Val Cys 35 40 45Leu Gly Gln
Asn Ser Gln Ser Pro Thr Ser Asn His Ser Pro Thr Ser 50 55 60Cys Pro
Pro Ile Cys Pro Gly Tyr Arg Trp Met Cys Leu Arg Arg Phe65 70 75
80Ile Ile Phe Leu Phe Ile Leu Leu Leu Cys Leu Ile Phe Leu Leu Val
85 90 95Leu Leu Asp Tyr Gln Gly Met Leu Pro Val Cys Pro Leu Ile Pro
Gly 100 105 110Ser Thr Thr Thr Ser Thr Gly Pro Cys Lys Thr Cys Thr
Thr Pro Ala 115 120 125Gln Gly Asn Ser Met Phe Pro Ser Cys Cys Cys
Thr Lys Pro Thr Asp 130 135 140Gly Asn Cys Thr Cys Ile Pro Ile Pro
Ser Ser Trp Ala Phe Ala Lys145 150 155 160Tyr Leu Trp Glu Trp Ala
Ser Val Arg Phe Ser Trp Leu Ser Leu Leu 165 170 175Val Pro Phe Val
Gln Trp Phe Val Gly Leu Ser Pro Thr Val Trp Leu 180 185 190Ser Ala
Ile Trp Met Met Trp Tyr Trp Gly Pro Ser Leu Tyr Ser Ile 195 200
205Val Ser Pro Phe Ile Pro Leu Leu Pro Ile Phe Phe Cys Leu Trp Val
210 215 220Tyr Ile225151467DNAHomo sapiens 15cgcagttgtg cacaccccag
cccaggctct ggcaggctgc caggcggggc tcgccgcctt 60cgcagtgcac gttgtccagc
aggatgtgtc cggtgccata gccgaagaag gcgttggtgg 120tggcggccat
ggccgccccg cagcccagct gacgacagac cacagcggca tccggcagcc
180cccagtcgtc gtcacacacg gtgccccaca ggccactgtg caggatctcc
actcggccct 240gacacagggt ggagccagcc atcctgagac gtccgtacag
cccgtactgg tggagtgtca 300ggaggccact ctgatggtca tggtcagcaa
agaccttttt ggcaccggga agctcatcag 360ggctgctgac ctcaccttgg
gcccagaggc ctgtgagcct ctggtctcca tggacacaga 420agatgtggtc
aggtttgagg ttggactcca cgagtgtggc aacagcatgc aggtaactga
480cgatgccctg gtgtacagca ccttcctgct ccatgacccc cgccccgtgg
gaaacctgtc 540catcgtgagg actaaccgcg cagagattcc catcgagtgc
cgctacccca ggcagggcaa 600tgtgagcagc caggccatcc tgcccacctg
gttgcccttc aggaccacgg tgttctcaga 660ggagaagctg actttctctc
tgcgtctgat ggaggagaac tggaacgctg agaagaggtc 720ccccaccttc
cacctgggag atgcagccca cctccaggca gaaatccaca ctggcagcca
780cgtgccactg cggttgtttg tggaccactg cgtggccaca ccgacaccag
accagaatgc 840ctccccttat cacaccatcg tggacttcca tggctgtctt
gtcgacggtc tcactgatgc 900ctcttctgca ttcaaagttc ctcgacccgg
gccagataca ctccagttca cagtggatgt 960cttccacttt gctaatgact
ccagaaacat gatatacatc acctgccacc tgaaggtcac 1020cctagctgag
caggacccag atgaactcaa caaggcctgt tccttcagca agccttccaa
1080cagctggttc ccagtggaag gctcggctga catctgtcaa tgctgtaaca
aaggtgactg 1140tggcactcca agccattcca ggaggcagcc tcatgtcatg
agccagtggt ccaggtctgc 1200ttcccgtaac cgcaggcatg tgacagaaga
agcagatgtc accgtggggc cactgatctt 1260cctggacagg aggggtgacc
atgaagtaga gcagtgggct ttgccttctg acacctcagt 1320ggtgctgctg
ggcgtaggcc tggctgtggt ggtgtccctg actctgactg ctgttatcct
1380ggttctcacc aggaggtgtc gcactgcctc ccaccctgtg tctgcttccg
aataaaagaa 1440gaaagcaata aaaaaaaaaa aaaaaaa 146716373PRTHomo
sapiens 16Met Val Met Val Ser Lys Asp Leu Phe Gly Thr Gly Lys Leu
Ile Arg1 5 10 15Ala Ala Asp Leu Thr Leu Gly Pro Glu Ala Cys Glu Pro
Leu Val Ser 20 25 30Met Asp Thr Glu Asp Val Val Arg Phe Glu Val Gly
Leu His Glu Cys 35 40 45Gly Asn Ser Met Gln Val Thr Asp Asp Ala Leu
Val Tyr Ser Thr Phe 50 55 60Leu Leu His Asp Pro Arg Pro Val Gly Asn
Leu Ser Ile Val Arg Thr65 70 75 80Asn Arg Ala Glu Ile Pro Ile Glu
Cys Arg Tyr Pro Arg Gln Gly Asn 85 90 95Val Ser Ser Gln Ala Ile Leu
Pro Thr Trp Leu Pro Phe Arg Thr Thr 100 105 110Val Phe Ser Glu Glu
Lys Leu Thr Phe Ser Leu Arg Leu Met Glu Glu 115 120 125Asn Trp Asn
Ala Glu Lys Arg Ser Pro Thr Phe His Leu Gly Asp Ala 130 135 140Ala
His Leu Gln Ala Glu Ile His Thr Gly Ser His Val Pro Leu Arg145 150
155 160Leu Phe Val Asp His Cys Val Ala Thr Pro Thr Pro Asp Gln Asn
Ala 165 170 175Ser Pro Tyr His Thr Ile Val Asp Phe His Gly Cys Leu
Val Asp Gly 180 185 190Leu Thr Asp Ala Ser Ser Ala Phe Lys Val Pro
Arg Pro Gly Pro Asp 195 200 205Thr Leu Gln Phe Thr Val Asp Val Phe
His Phe Ala Asn Asp Ser Arg 210 215 220Asn Met Ile Tyr Ile Thr Cys
His Leu Lys Val Thr Leu Ala Glu Gln225 230 235 240Asp Pro Asp Glu
Leu Asn Lys Ala Cys Ser Phe Ser Lys Pro Ser Asn 245 250 255Ser Trp
Phe Pro Val Glu Gly Ser Ala Asp Ile Cys Gln Cys Cys Asn 260 265
270Lys Gly Asp Cys Gly Thr Pro Ser His Ser Arg Arg Gln Pro His Val
275 280 285Met Ser Gln Trp Ser Arg Ser Ala Ser Arg Asn Arg Arg His
Val Thr 290 295 300Glu Glu Ala Asp Val Thr Val Gly Pro Leu Ile Phe
Leu Asp Arg Arg305 310 315 320Gly Asp His Glu Val Glu Gln Trp Ala
Leu Pro Ser Asp Thr Ser Val 325 330 335Val Leu Leu Gly Val Gly Leu
Ala Val Val Val Ser Leu Thr Leu Thr 340 345 350Ala Val Ile Leu Val
Leu Thr Arg Arg Cys Arg Thr Ala Ser His Pro 355 360 365Val Ser Ala
Ser Glu 370171740DNAAvian influenza virus 17atggaaagaa tagtgattgc
ccttgcaata atcaacattg tcaaaggtga ccaaatctgc 60attggttatc atgcaaacaa
ttcaacagag caggttgaca caatcatgga gaagaatgtg 120acggtcacac
atgctcagga catactggaa aaagagcaca atgggaaact ctgcagtctt
180aaaggagtga ggcccctcat tctgaaggat tgcagtgtcg ctgggtggct
tcttggaaac 240ccaatgtgtg atgaattcct gaatgtaccg gaatggtcat
acattgtgga aaaagataat 300ccagtcaatg gcctgtgcta tccaggagac
ttcaacgatt atgaagaact gaagcattta 360atgagcagca caaaccattt
tgagaaaatt cagataattc ctaggaactc ttggtccacc 420catgatgcct
catcaggagt gagctcagca tgcccataca atggtaggtc ttcctttttc
480aggaatgtag tgtggttgat caagaagaat aatgcgtacc caacaataaa
gaggacctac 540aacaacacca atgtagaaga ccttttaata ttatggggaa
tccaccaccc taatgatgca 600gcagaacaaa caaaactcta ccagaactcg
aacacttatg tgtctgtagg aacatcaaca 660ctgaatcaga ggtcaatccc
agaaatagcc accagaccca aagtgaacgg acaaagtgga 720agaatggaat
ttttttggac aatactaagg ccgaacgatg caatcagctt tgaaagtaat
780gggaacttta tagctcctga atatgcgtac aagattgtta aaaaaggaga
ttcagcaatc 840atgaaaagtg aactggagta tggtaactgt gataccaaat
gtcagacccc agtgggtgct 900ataaattcca gtatgccttt ccacaatgtt
catcccctta ccattgggga gtgccccaag 960tatgtcaaat cggacaaact
ggtccttgca acaggactaa gaaacgtacc ccaaagagaa 1020acaagaggcc
tatttggagc aatagcagga ttcatagaag gaggatggca aggaatggta
1080gatggatggt atggatacca tcatagcaat gagcagggaa gtggatatgc
tgcagacaaa 1140gaatctaccc agaaagcaat cgatgggatc accaataaag
taaactcaat cattgacaaa 1200atgaacactc aattcgaagc cgttgggaaa
gaattcaaca acctagaaag gagaatagaa 1260aatttgaata agaaaatgga
agatgggttt ttagatgtat ggacttacaa tgcagaactt 1320ctagtgctca
tggaaaacga aagaactctg gatttccatg attcaaatgt caagaactta
1380tacgataagg tccgactcca gctgagagac aatgcaaaag aattaggcaa
cgggtgcttt 1440gaattctacc acaagtgtga caatgaatgc atggaaagtg
tgagaaatgg aacgtatgac 1500tatccacaat actcagaaga atcaagactg
aacagggagg aaatagacgg agtcaaattg 1560gaatcaatgg gcacttatca
gatactatca atctactcaa cagtggcgag ttccctagca 1620ctggcaatca
tggtagctgg tctatctttt tggatgtgct ccaatggatc attgcagtgc
1680agaatttgca tctagaattg tgagttcaga ttataattaa aaacacccta
gtttctactg 174018564PRTAvian influenza virus 18Met Glu Arg Ile Val
Ile Ala Leu Ala Ile Ile Asn Ile Val Lys Gly1 5 10 15Asp Gln Ile Cys
Ile Gly Tyr His Ala Asn Asn Ser Thr Glu Gln Val 20 25 30Asp Thr Ile
Met Glu Lys Asn Val Thr Val Thr His Ala Gln Asp Ile 35 40 45Leu Glu
Lys Glu His Asn Gly Lys Leu Cys Ser Leu Lys Gly Val Arg 50 55 60Pro
Leu Ile Leu Lys Asp Cys Ser Val Ala Gly Trp Leu Leu Gly Asn65 70 75
80Pro Met Cys Asp Glu Phe Leu Asn Val Pro Glu Trp Ser Tyr Ile Val
85 90 95Glu Lys Asp Asn Pro Val Asn Gly Leu Cys Tyr Pro Gly Asp Phe
Asn 100 105 110Asp Tyr Glu Glu Leu Lys His Leu Met Ser Ser Thr Asn
His Phe Glu 115 120 125Lys Ile Gln Ile Ile Pro Arg Asn Ser Trp Ser
Thr His Asp Ala Ser 130 135 140Ser Gly Val Ser Ser Ala Cys Pro Tyr
Asn Gly Arg Ser Ser Phe Phe145 150 155 160Arg Asn Val Val Trp Leu
Ile Lys Lys Asn Asn Ala Tyr Pro Thr Ile 165 170 175Lys Arg Thr Tyr
Asn Asn Thr Asn Val Glu Asp Leu Leu Ile Leu Trp 180 185 190Gly Ile
His His Pro Asn Asp Ala Ala Glu Gln Thr Lys Leu Tyr Gln 195 200
205Asn Ser Asn Thr Tyr Val Ser Val Gly Thr Ser Thr Leu Asn Gln Arg
210 215 220Ser Ile Pro Glu Ile Ala Thr Arg Pro Lys Val Asn Gly Gln
Ser Gly225 230 235 240Arg Met Glu Phe Phe Trp Thr Ile Leu Arg Pro
Asn Asp Ala Ile Ser 245 250 255Phe Glu Ser Asn Gly Asn Phe Ile Ala
Pro Glu Tyr Ala Tyr Lys Ile 260 265 270Val Lys Lys Gly Asp Ser Ala
Ile Met Lys Ser Glu Leu Glu Tyr Gly 275 280 285Asn Cys Asp Thr Lys
Cys Gln Thr Pro Val Gly Ala Ile Asn Ser Ser 290 295 300Met Pro Phe
His Asn Val His Pro Leu Thr Ile Gly Glu Cys Pro Lys305 310 315
320Tyr Val Lys Ser Asp Lys Leu Val Leu Ala Thr Gly Leu Arg Asn Val
325 330 335Pro Gln Arg Glu Thr Arg Gly Leu Phe Gly Ala Ile Ala Gly
Phe Ile 340 345 350Glu Gly Gly Trp Gln Gly Met Val Asp Gly Trp Tyr
Gly Tyr His His 355 360 365Ser Asn Glu Gln Gly Ser Gly Tyr Ala Ala
Asp Lys Glu Ser Thr Gln 370 375 380Lys Ala Ile Asp Gly Ile Thr Asn
Lys Val Asn Ser Ile Ile Asp Lys385 390 395 400Met Asn Thr Gln Phe
Glu Ala Val Gly Lys Glu Phe Asn Asn Leu Glu 405 410 415Arg Arg Ile
Glu Asn Leu Asn Lys Lys Met Glu Asp Gly Phe Leu Asp 420 425 430Val
Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Met Glu Asn Glu Arg 435 440
445Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Asp Lys Val
450 455 460Arg Leu Gln Leu Arg Asp Asn Ala Lys Glu Leu Gly Asn Gly
Cys Phe465 470 475 480Glu Phe Tyr His Lys Cys Asp Asn Glu Cys Met
Glu Ser Val Arg Asn 485 490 495Gly Thr Tyr Asp Tyr Pro Gln Tyr Ser
Glu Glu Ser Arg Leu Asn Arg 500 505 510Glu Glu Ile Asp Gly Val Lys
Leu Glu Ser Met Gly Thr Tyr Gln Ile 515 520 525Leu Ser Ile Tyr Ser
Thr Val Ala Ser Ser Leu Ala Leu Ala Ile Met 530 535 540Val Ala Gly
Leu Ser Phe Trp Met Cys Ser Asn Gly Ser Leu Gln Cys545 550 555
560Arg Ile Cys Ile191734DNANewcastle disease virus 19atggaccgcg
ccgttagcca agttgcgtta gagaatgatg aaagagaggc aaaaaataca 60tggcgcttga
tattccggat tgcaatctta ttcttaacag tagtgacctt ggctatatct
120gtagcctccc ttttatatag catgggggct agcacaccta gcgatcttgt
aggcataccg 180actaggattt ccagggcaga agaaaagatt acatctacac
ttggttccaa tcaagatgta 240gtagatagga tatataagca agtggccctt
gagtctccgt tggcattgtt aaaaactgag 300accacaatta tgaacgcaat
aacatctctc tcttatcaga ttaatggagc tgcaaacaac 360agcgggtggg
gggcacctat ccatgaccca gattatatag gggggatagg caaagaactc
420attgtagatg atgctagtga tgtcacatca ttctatccct ctgcatttca
agaacatctg 480aattttatcc cggcgcctac tacaggatca ggttgcactc
gaataccctc atttgacatg 540agtgctaccc attactgcta cacccataat
gtaatattgt ctggatgcag agatcactca 600cattcatatc agtatttagc
acttggtgtg ctccggacat ctgcaacagg gggggtattc 660ttttctactc
tgcgttccat caacctggac gacacccaaa atcggaagtc ttgcagtgtg
720agtgcaactc ccctgggttg tgatatgctg tgctcgaaag tcacggagac
agaggaagaa 780gattataact cagctgtccc tacgcggatg gtacatggga
ggttagggtt cgacggccag 840taccacgaaa aggacctaga tgtcacaaca
ttattcgggg actgggtggc caactaccca 900ggagtagggg gtggatcttt
tattgacagc cgcgtatggt tctcagttta cggagggtta 960aaacccaatt
cacccagtga cactgtacag gaagggaaat atgtgatata caagcaatac
1020aatgacacat gcccagatga gcaagactac cagattcgaa tggccaagtc
ttcgtataag 1080cctggacggt ttggtgggaa acgcatacag caggctatct
tatctatcaa ggtgtcaaca 1140tccttaggcg aagacccggt actgactgta
ccgcccaaca cagtcacact catgggggcc 1200gaaggcagaa ttctcacagt
agggacatct catttcttgt atcaacgagg gtcatcatac 1260ttctctcccg
cgttattata tcctatgaca gtcagcaaca aaacagccac tcttcatagt
1320ccttatacat tcaatgcctt cactcggcca ggtagtatcc cttgccaggc
ttcagcaaga 1380tgccccaacc cgtgtgttac tggagtctat acagatccat
atcccctaat cttctataga 1440aaccacacct tgcgaggggt attcgggaca
atgcttgatg gtgtacaagc aagacttaac 1500cctgcgtctg cagtattcga
tagcacatcc cgcagtcgca ttactcgagt gagttcaagc 1560agtaccaaag
cagcatacac aacatcaact tgttttaaag tggtcaagac taataagacc
1620tattgtctca gcattgctga aatatctaat actctcttcg gagaattcag
aatcgtcccg 1680ttactagttg agatcctcaa agatgacggg gttagagaag
ccaggtctgg ctag 173420577PRTNewcastle disease virus 20Met Asp Arg
Ala Val Ser Gln Val Ala Leu Glu Asn Asp Glu Arg Glu1 5 10 15Ala Lys
Asn Thr Trp Arg Leu Ile Phe Arg Ile Ala Ile Leu Phe Leu 20 25 30Thr
Val Val Thr Leu Ala Ile Ser Val Ala Ser Leu Leu Tyr Ser Met 35 40
45Gly Ala Ser Thr Pro Ser Asp Leu Val Gly Ile Pro Thr Arg Ile Ser
50 55 60Arg Ala Glu Glu Lys Ile Thr Ser Thr Leu Gly Ser Asn Gln Asp
Val65 70 75 80Val Asp Arg Ile Tyr Lys Gln Val Ala Leu Glu Ser Pro
Leu Ala Leu 85 90 95Leu Lys Thr Glu Thr Thr Ile Met Asn Ala Ile Thr
Ser Leu Ser Tyr 100 105 110Gln Ile Asn Gly Ala Ala Asn Asn Ser Gly
Trp Gly Ala Pro Ile His 115 120 125Asp Pro Asp Tyr Ile Gly Gly Ile
Gly Lys Glu Leu Ile Val Asp Asp 130 135 140Ala Ser Asp Val Thr Ser
Phe Tyr Pro Ser Ala Phe Gln Glu His Leu145 150 155 160Asn Phe Ile
Pro Ala Pro Thr Thr Gly Ser Gly Cys Thr Arg Ile Pro 165 170 175Ser
Phe Asp Met Ser Ala Thr His Tyr Cys Tyr Thr His Asn Val Ile 180
185 190Leu Ser Gly Cys Arg Asp His Ser His Ser Tyr Gln Tyr Leu Ala
Leu 195 200 205Gly Val Leu Arg Thr Ser Ala Thr Gly Gly Val Phe Phe
Ser Thr Leu 210 215 220Arg Ser Ile Asn Leu Asp Asp Thr Gln Asn Arg
Lys Ser Cys Ser Val225 230 235 240Ser Ala Thr Pro Leu Gly Cys Asp
Met Leu Cys Ser Lys Val Thr Glu 245 250 255Thr Glu Glu Glu Asp Tyr
Asn Ser Ala Val Pro Thr Arg Met Val His 260 265 270Gly Arg Leu Gly
Phe Asp Gly Gln Tyr His Glu Lys Asp Leu Asp Val 275 280 285Thr Thr
Leu Phe Gly Asp Trp Val Ala Asn Tyr Pro Gly Val Gly Gly 290 295
300Gly Ser Phe Ile Asp Ser Arg Val Trp Phe Ser Val Tyr Gly Gly
Leu305 310 315 320Lys Pro Asn Ser Pro Ser Asp Thr Val Gln Glu Gly
Lys Tyr Val Ile 325 330 335Tyr Lys Gln Tyr Asn Asp Thr Cys Pro Asp
Glu Gln Asp Tyr Gln Ile 340 345 350Arg Met Ala Lys Ser Ser Tyr Lys
Pro Gly Arg Phe Gly Gly Lys Arg 355 360 365Ile Gln Gln Ala Ile Leu
Ser Ile Lys Val Ser Thr Ser Leu Gly Glu 370 375 380Asp Pro Val Leu
Thr Val Pro Pro Asn Thr Val Thr Leu Met Gly Ala385 390 395 400Glu
Gly Arg Ile Leu Thr Val Gly Thr Ser His Phe Leu Tyr Gln Arg 405 410
415Gly Ser Ser Tyr Phe Ser Pro Ala Leu Leu Tyr Pro Met Thr Val Ser
420 425 430Asn Lys Thr Ala Thr Leu His Ser Pro Tyr Thr Phe Asn Ala
Phe Thr 435 440 445Arg Pro Gly Ser Ile Pro Cys Gln Ala Ser Ala Arg
Cys Pro Asn Pro 450 455 460Cys Val Thr Gly Val Tyr Thr Asp Pro Tyr
Pro Leu Ile Phe Tyr Arg465 470 475 480Asn His Thr Leu Arg Gly Val
Phe Gly Thr Met Leu Asp Gly Val Gln 485 490 495Ala Arg Leu Asn Pro
Ala Ser Ala Val Phe Asp Ser Thr Ser Arg Ser 500 505 510Arg Ile Thr
Arg Val Ser Ser Ser Ser Thr Lys Ala Ala Tyr Thr Thr 515 520 525Ser
Thr Cys Phe Lys Val Val Lys Thr Asn Lys Thr Tyr Cys Leu Ser 530 535
540Ile Ala Glu Ile Ser Asn Thr Leu Phe Gly Glu Phe Arg Ile Val
Pro545 550 555 560Leu Leu Val Glu Ile Leu Lys Asp Asp Gly Val Arg
Glu Ala Arg Ser 565 570 575Gly211377DNAGallus gallus 21atgctgggtg
agctggaagc aggcgggatg caaggcggcc gcgtggtgct ggggctcctg 60tgctgcttgg
tggccggggt gggctcctac acgccctggg acatctcctg ggcagcacga
120ggggacccct ctgcctggtc ctggggggcc gaggcgcact cacgggccgt
ggccggctcg 180cacccggtgg ccgtgcagtg ccaagaggcg cagctggtgg
tgacggtgca cagggacctc 240ttcgggaccg ggcgtctcat caacgctgct
gacctgactc tgggcccggc tgcctgcaag 300cactcctcgc tcaacgccgc
acacaacacc gtcaccttcg ccgccggcct ccacgagtgc 360ggcagcgtcg
tgcaggtgac gccagacacc ctcatctacc gcacgctcat caactacgac
420cccagccctg ctagcaaccc cgtcatcatc cgcaccaacc ctgctgtcat
ccccatcgag 480tgccactacc ccaggaggga gaacgtgagc agcaatgcca
tccggcccac ctggtccccc 540ttcaactccg cactgtcagc cgaggagagg
ctggtgttct ccctgcgcct catgagtgat 600gactggagca cagagagacc
cttcaccggc ttccagctgg gcgacatcct caacatccag 660gccgaggtca
gcactgagaa ccatgtgccc ctgcggctct ttgtggacag ctgtgtggct
720gccctgagcc ctgacggtga ctcctcgccc cactacgcca tcattgactt
caacgggtgc 780ttagtggatg ggagagtgga tgatactagc tctgccttca
tcacaccccg gccacgggag 840gatgtgctga ggttcaggat cgatgtcttc
aggtttgcgg gggacaacag gaacctgatc 900tacatcacct gccacctgaa
ggtgacccca gcagaccaag gcccagaccc tcagaacaag 960gcttgctcct
tcaataaagc cagaaacacc tgggtgccag tggaaggcag ccgggatgtc
1020tgcaactgct gtgagacagg caactgcgag ccgcctgcgc tctcccggag
gctcaacccc 1080atggagagat ggcagagccg ccgcttccgt cgtgatgccg
ggaaagaggt tgcagctgat 1140gtggtcattg gccccgtgtt gctctcggcg
gacccgggag ctgtgggaca gcaggaggag 1200ggtggtgacg gtgcggcggt
gatggtgccc agcgtgggga cggggctggt gtgcgtggcc 1260gtggctgtag
ctctggctgc cgttggggtg gctgtatgta ttgcacgcaa gggatgcacc
1320cgaacctcaa ctgcggtgtg agtgcagggc gagccgtgaa taaagcctgg aaaggcc
137722446PRTGallus gallus 22Met Leu Gly Glu Leu Glu Ala Gly Gly Met
Gln Gly Gly Arg Val Val1 5 10 15Leu Gly Leu Leu Cys Cys Leu Val Ala
Gly Val Gly Ser Tyr Thr Pro 20 25 30Trp Asp Ile Ser Trp Ala Ala Arg
Gly Asp Pro Ser Ala Trp Ser Trp 35 40 45Gly Ala Glu Ala His Ser Arg
Ala Val Ala Gly Ser His Pro Val Ala 50 55 60Val Gln Cys Gln Glu Ala
Gln Leu Val Val Thr Val His Arg Asp Leu65 70 75 80Phe Gly Thr Gly
Arg Leu Ile Asn Ala Ala Asp Leu Thr Leu Gly Pro 85 90 95Ala Ala Cys
Lys His Ser Ser Leu Asn Ala Ala His Asn Thr Val Thr 100 105 110Phe
Ala Ala Gly Leu His Glu Cys Gly Ser Val Val Gln Val Thr Pro 115 120
125Asp Thr Leu Ile Tyr Arg Thr Leu Ile Asn Tyr Asp Pro Ser Pro Ala
130 135 140Ser Asn Pro Val Ile Ile Arg Thr Asn Pro Ala Val Ile Pro
Ile Glu145 150 155 160Cys His Tyr Pro Arg Arg Glu Asn Val Ser Ser
Asn Ala Ile Arg Pro 165 170 175Thr Trp Ser Pro Phe Asn Ser Ala Leu
Ser Ala Glu Glu Arg Leu Val 180 185 190Phe Ser Leu Arg Leu Met Ser
Asp Asp Trp Ser Thr Glu Arg Pro Phe 195 200 205Thr Gly Phe Gln Leu
Gly Asp Ile Leu Asn Ile Gln Ala Glu Val Ser 210 215 220Thr Glu Asn
His Val Pro Leu Arg Leu Phe Val Asp Ser Cys Val Ala225 230 235
240Ala Leu Ser Pro Asp Gly Asp Ser Ser Pro His Tyr Ala Ile Ile Asp
245 250 255Phe Asn Gly Cys Leu Val Asp Gly Arg Val Asp Asp Thr Ser
Ser Ala 260 265 270Phe Ile Thr Pro Arg Pro Arg Glu Asp Val Leu Arg
Phe Arg Ile Asp 275 280 285Val Phe Arg Phe Ala Gly Asp Asn Arg Asn
Leu Ile Tyr Ile Thr Cys 290 295 300His Leu Lys Val Thr Pro Ala Asp
Gln Gly Pro Asp Pro Gln Asn Lys305 310 315 320Ala Cys Ser Phe Asn
Lys Ala Arg Asn Thr Trp Val Pro Val Glu Gly 325 330 335Ser Arg Asp
Val Cys Asn Cys Cys Glu Thr Gly Asn Cys Glu Pro Pro 340 345 350Ala
Leu Ser Arg Arg Leu Asn Pro Met Glu Arg Trp Gln Ser Arg Arg 355 360
365Phe Arg Arg Asp Ala Gly Lys Glu Val Ala Ala Asp Val Val Ile Gly
370 375 380Pro Val Leu Leu Ser Ala Asp Pro Gly Ala Val Gly Gln Gln
Glu Glu385 390 395 400Gly Gly Asp Gly Ala Ala Val Met Val Pro Ser
Val Gly Thr Gly Leu 405 410 415Val Cys Val Ala Val Ala Val Ala Leu
Ala Ala Val Gly Val Ala Val 420 425 430Cys Ile Ala Arg Lys Gly Cys
Thr Arg Thr Ser Thr Ala Val 435 440 445233024DNAAnas sp.
23catgcacacc tgaaagctta tgcaaagatt aacgaggaat cactggatag ggctaggaga
60ttgctttggt ggcattacaa ttgtttactg tggggagaag ctaacgttac taattatatt
120tctcggcttc gcacttggct atcaacacct gagagataca gaggccgaga
tgccccaacc 180attgaagcaa tcactagacc aatccaagtg gctcagggag
gcagaaaaac atcttcgggt 240actagaaaac ctcgtggact cgaacctaga
agaagaaaag ttaaaaccac agttgtctat 300gggagaagac gttcaaagtc
cagggatagg agagcccctt caccccaacg tgcgggctcc 360cctctcccgc
gtagttcgag cagccacaga agatctccct cgcctaggaa atagattacc
420tgctaggcat caccttggta aattgtcagg attatatcaa atgaagggat
gtacatttaa 480ccctgaatgg aaagtacctg atatttcgga tactcatttt
gatatgcaaa tagtaaatga 540gtgcccttcc cgaaattgga aatatctgac
tccagccaaa ttctggccca agagcatttc 600ctactttcct gtacaggcag
gggttaaagc taagtaccct gacaatgtga tgcaacatga 660atcaatagta
ggtaaatatt taaccaggct ctatgaagca ggaatccttt ataagcggat
720atctaaacat ttggtcacat ttaaaggtca gccttataat tgggaacttc
aataccttgt 780caagcaacat caagttcctg atgggtcaac aacctgcaaa
atcaatggac gtgcggagaa 840tcgaaggagg agaactcctg ctaaatcaat
tagcaggccg catgatccca aaagggacag 900tcacatggtc gggcaaattt
ccaacaatag atcacatatt agaccatgtg caaacaatgg 960aggaaataaa
cactcttcaa aaccaagggg cttggcctgc tggggcggga aggagagcag
1020gattaaccaa tcctgctcct caagagattc ctcagcccca gtggactccc
gaagaagatc 1080agaaagctcg cgaagctttt cgtcgttatc aagaagagag
accaccagag accaccacaa 1140ttcctcccac atctccaacg cagtggaaac
tgcaacccgg ggacgatcca ctcctgggaa 1200acaagtctct gctcgagact
cacccgcttt accagaatac cgagccagcc gtgtctgtaa 1260taaagactcc
tccactgaga aagaaaatgt ctggtacctt cgggggaata ctagctggcc
1320taatcggatt actggtaagc tttttcttgt tgataaaaat tctagaaata
cttcggaggc 1380tagattggtg gtggatttct ctcagttctc caaagggaaa
aatgcaatgc gctttccaag 1440atactggagc ccaaacctct ccacattacg
tcggatcttg cccgtgggga tgcccaggat 1500ttctctggac ttatctcagg
ctttttatca tcttcctctt aatcctgcta gtagcagcag 1560gcttgctgta
tctgacggac aacgggtcta ctattttagg aaagctccga tgggagtcgg
1620tcttagccct ttcctcctcc atctcttcac tactgccctc ggatccgaaa
tcgctcgtcg 1680ctttaatgtt tggactttta cttatatgga tgacttcctc
ctctgccacc caaacgctcg 1740tcaccttaac tcaattagcc acgctgtctg
ctctttttta caagagctag gaataagaat 1800aaactttgac aaaactactc
catcaccagt caacgaaatt agattcctcg gttatcaaat 1860tgatcaacga
ttcatgaaga ttgaagaaag cagatggaaa gaattacgga ctgtaattaa
1920aaagataaaa attggagaat ggtatgactg gaaatgtatt cagagatttg
tcgggcattt 1980aaactttgtg ttgccattta ccaaaggtaa catagaaatg
ttaaaaccaa tgtatgctgc 2040tataactcat aaagtcaatt ttagcttctc
ttctgcctat aggactttgc tgtacaaatt 2100aactatgggt gtttgtaaat
tatcaatcaa accaaagtcc tctgtacctt tgccacgtgt 2160agctacggat
gctaccccaa cacatggcgc aatatcccat atcaccggcg ggagcgcagt
2220gtttgctttt tcaaaggtca gagatataca tatacaggaa ttgctgatgg
tatgtttagc 2280taaattaatg attaagccta gatgcatact aaccgattct
acctttgttt gtcacaaacg 2340ttatcagacg ttaccatgga attttgcagt
gtttgccaaa caattgttat cttctatacc 2400attgtacttt gtaccgagca
aatataatcc tgctgacggc ccatccaggc acaaaccgcc 2460tgattggacg
gctgttacat acacccctct ctcgaaagca atatatattc cacataggct
2520atgtggaact taagaattac acccctctcc ttcggagctg cctgccaagg
tatctttacg 2580tctacattgc tgttgtcgtg tttgactgta cctttggtat
gtaccattgt ttatgattct 2640tgcttatata tggatatcaa tgcttctaga
gccttagcca atgtttatga tttgccagat 2700gatttcttcc caaaaattga
tgatcttgta agggatgcga aggatgcttt agaaccttat 2760tggagatcag
attcaataaa gaaacatgtt ttaattgcaa ctcactttgt ggatcttatt
2820gaagacttct ggcaaactac tcagggtatg catgaaatag ctgaagcctt
aagagcagtt 2880ataccaccta ctacaacacc agttcccgca ggatatctga
ttcagcacga agaggctgag 2940gagattcctc tgggagattt atttaaacat
caggaagaaa ggatagttag tttccaaccg 3000gattatccta ttactgcacg aatt
30242410575DNAArtificial SequencepCHA vector sequence 24ggccgcaaca
gaggtggatg gacagacccg ttcttacacc ggactgggcg cgggatagga 60tattcagatt
gggatgggat tgagcttaaa gccggcgctg agaccatgct caaggtaggc
120aatgtcctca gcgtcgagcc cggcatctat gtcgagggca ttggtggagc
gcgcttcggg 180gataccgtgc ttgtaactga gaccggatat gaggccctca
ctccgcttga tcttggcaaa 240gatatttgac gcatttatta gtatgtgtta
attttcattt gcagtgcagt attttctatt 300cgatctttat gtaattcgtt
acaattaata aatattcaaa tcagattatt gactgtcatt 360tgtatcaaat
cgtgtttaat ggatattttt attataatat tgatgatatc tcaatcaaaa
420cgtagataat aataatattt atttaatatt tttgcgtcgc acagtgaaaa
tctatatgag 480attacaaaat accgacaaca ttatttaaga tacatagaca
ttaaccctga gactgttgga 540cagagctcat tggtacctca gatctgggta
actggcctaa ctggccttgg aggagctggc 600aactcaaaat ccctttgcca
aaaaccaaca tcatgccatc caccatgctt gtatccagct 660gcgcgcaatg
taccccgggc tgtgtatccc aaagcctcat gcaacctaac agatggatcg
720tttggaaggc ctataacagc aaccacagac ttaaaacctt gcgcctccat
agacttaagc 780aaatgtgtgt acaatgtgga tcctaggccc aacctttgat
gcctatgtga cacgtaaaca 840gtactctcaa ctgtccaatc gtaagcgttc
ctagccttcc agggcccagc gtaagcaata 900ccagccacaa caccctcaac
ctcagcaacc aaccaagggt atctatcttg caacctctct 960agatcatcaa
tccactcttg tggtgtttgt ggctctgtcc taaagttcac tgtagacgtc
1020tcaatgtaat ggttaacgat atcacaaacc gcggccatat cagctgctgt
agctggccta 1080atctcaactg gtctcctctc cggagaagcc atggtttgga
tccacaaact tacaaatttc 1140tctgaagttg tatcctcagt acttcaaaga
aaatagctta caccaatttt ttcttgtttt 1200cacaaatgcc gaacttggtt
ccttatatag gaaaactcaa gggcaaaaat gacacggaaa 1260aatataaaag
gataagtagt gggggataag attcctttgt gataaggtta ctttccgccc
1320ttacattttc caccttacat gtgtcctcta tgtctctttc acaatcaccg
accttatctt 1380cttcttttca ttgttgtcgt cagtgcttac gtcttcaaga
ttcttttctt cgcctggttc 1440ttctttttca atttctacgt attcttcttc
gtattctggc agtataggat cttgtatctg 1500tacattcttc atttttgaac
ataggttgca tatgtgccgc atattgatct gcttcttgct 1560gagctcacat
aatacttcca tagtttttcc cgtaaacatt ggattcttga tgctacatct
1620tggataatta ccttctggcc ggccgcgaat tcgcttcaag acgtgctcaa
atcactattt 1680ccacacccct atatttctat tgcactccct tttaactgtt
ttttattaca aaaatgccct 1740ggaaaatgca ctcccttttt gtgtttgtta
tttagtgaaa cgatgttgtc aggtaattta 1800tttgtcagtc tactatggtg
gcccattata ttaatagcaa ctgtcggtcc aatagacgac 1860gtcgattttc
tgcatttgtt taaccacgtg gattttatga cattttatat tagttaattt
1920gtaaaaccta cccaattaaa gacctcatat gttctaaaga ctaatactta
atgataacaa 1980ttttctttta gtgaagaaag ggataattag taaatatgga
acaagggcag aagatttatt 2040aaagccggta agagacaaca acgtaggtac
gtggagtgtc ttaggtgact tacccacata 2100acataaagtg acattaacaa
acatagctaa tgctcctatt tgaatagtgc atatcagcat 2160accttattac
atatagatag gagcaaactc tagctagatt gttgagagag ctcggtacct
2220taaaatctga actcacaatc ctagatgcaa attctgcact gcaatgatcc
attggagcac 2280atccaaaaag acagaccagc taccatgatt gccagtgcta
gggaactcgc cactgttgag 2340tagattgata gtatctgata ggtgcccatt
gactccaatt tgactccatc tatttcctct 2400ctgttcagcc ttgattcttc
tgagtattgt ggatagtcat acgttccgtt tctcacactt 2460tccatgcatt
cattgtcaca tttgtggtag aactcaaaac acccattgcc caattctttt
2520gcattatctc tcagctggag tcggacctta tcgtataggt tcttgacata
tgaatcatgg 2580aaatccagag ttctttcatt ttccatgagc accagaagtt
ctgcattgta agtccataca 2640tctagaaatc catcttccat tttcttattc
aaattttcta ttctcctttc taagttgttg 2700aattctttcc caacggcttc
gaattgagtg ttcattttgt caatgattga gttgacttta 2760ttggtgatcc
cgtcgattgc tttctgagtg gactctttgt ctgcagcata tccacttccc
2820tgctcgttgc tatgatggta accataccat ccatctacca ttccttgcca
ccccccttct 2880atgaatcctg ctattgctcc aaacagacct cttgtttctc
tctgaggcac gttcctcagt 2940cctgttgcaa ggaccagttt atctgatttg
acatatttgg gacactctcc aatggtaagg 3000ggatgaacat tgtgaaaagg
catactggaa tttatagcac ccactggggt ctgacatttg 3060gtatcacagt
tgccatactc cagttcgctt ctcatgattg ctgaatctcc ctttttaact
3120atcttgtatg catattcagg agctataaag ttcccattac tttcaaagct
gattgcatcg 3180ttcggcctta gtattgtcca gaaaaattct attcttccac
tttgtccatt cactttgggc 3240ctggtagcta tttctggaat tgacctctga
tttagtgttg atgttcctac agacacataa 3300gtgttcgagt tctgatagag
ttccgtttgt tccgctgcat cattagggtg atggattccc 3360cacaatatca
gaaggtcctc tacattggtg ttattgtagg tcctctttat tgttgggtat
3420gcattactct tcttgatcaa ccacaccaca ttcctgaaaa aggaagatct
accattgtat 3480gggcatgctg agctcactcc tgatgaggca tcatgattgg
accaagagtt cctagggatt 3540atttgaattt tctcaaaatg gtttgtgttg
ctcattaaat acttcagttc ttcataatca 3600ttgaagtctc ccggataaca
taagccattg gttggattgt ccttctctac aatatatgac 3660cattccggta
catttaggaa ctcatcacac attgggttcc caagaagcca tccagccaca
3720ctgcaatcct tcagaatgag gggcctcact cctttgagac tgcagagttt
cccgttgtgc 3780tctttttcca gtatatcttg agcatgtgtg accgtaacat
tcttctccat gattgtgtca 3840acttgttttg ttgaattgtt tgcatgataa
ccgatgcaga tttggtcacc tttgacaacg 3900ctgattattg caagggcaat
cactattctt tccatccatg gtttggatcc acaaacttac 3960aaatttctct
gaagttgtat cctcagtact tcaaagaaaa tagcttacac caattttttc
4020ttgttttcac aaatgccgaa cttggttcct tatataggaa aactcaaggg
caaaaatgac 4080acggaaaaat ataaaaggat aagtagtggg ggataagatt
cctttgtgat aaggttactt 4140tccgccctta cattttccac cttacatgtg
tcctctatgt ctctttcaca atcaccgacc 4200ttatcttctt cttttcattg
ttgtcgtcag tgcttacgtc ttcaagattc ttttcttcgc 4260ctggttcttc
tttttcaatt tctacgtatt cttcttcgta ttctggcagt ataggatctt
4320gtatctgtac attcttcatt tttgaacata ggttgcatat gtgccgcata
ttgatctgct 4380tcttgctgag ctcacataat acttccatag tttttcccgt
aaacattgga ttcttgatgc 4440tacatcttgg ataattacct tctggcgcgc
ctttgcccgg gctttcctgc agggtttaaa 4500cttaattaag cggccgatcc
ggtgagtaat attgtacggc taagagcgaa tttggcctgt 4560agacctcaat
tgcgagcttt ctaatttcaa actattcggg cctaactttt ggtgtgatga
4620tgctgactgg caggatatat accgttgtaa tttgagctcg tgtgaataag
tcgctgtgta 4680tgtttgtttg attgtttctg ttggagtgca gcccatttca
ccggacaagt cggctagatt 4740gatttagccc tgatgaactg ccgaggggaa
gccatcttga gcgcggaatg ggaatggatc 4800gaaccgggag cacaggatga
cgcctaacaa ttcattcaag ccgacaccgc ttcgcggcgc 4860ggcttaattc
aggagttaaa catcatgagg gaagcggtga tcgccgaagt atcgactcaa
4920ctatcagagg tagttggcgt catcgagcgc catctcgaac cgacgttgct
ggccgtacat 4980ttgtacggct ccgcagtgga tggcggcctg aagccacaca
gtgatattga tttgctggtt 5040acggtgaccg taaggcttga tgaaacaacg
cggcgagctt tgatcaacga ccttttggaa 5100acttcggctt cccctggaga
gagcgagatt ctccgcgctg tagaagtcac cattgttgtg 5160cacgacgaca
tcattccgtg gcgttatcca gctaagcgcg aactgcaatt tggagaatgg
5220cagcgcaatg acattcttgc aggtatcttc gagccagcca cgatcgacat
tgatctggct 5280atcttgctga caaaagcaag agaacatagc gttgccttgg
taggtccagc ggcggaggaa 5340ctctttgatc cggttcctga acaggatcta
tttgaggcgc taaatgaaac cttaacgcta 5400tggaactcgc
cgcccgactg ggctggcgat gagcgaaatg tagtgcttac gttgtcccgc
5460atttggtaca gcgcagtaac cggcaaaatc gcgccgaagg atgtcgctgc
cgactgggca 5520atggagcgcc tgccggccca gtatcagccc gtcatacttg
aagctaggca ggcttatctt 5580ggacaagaag atcgcttggc ctcgcgcgca
gatcagttgg aagaatttgt tcactacgtg 5640aaaggcgaga tcaccaaggt
agtcggcaaa taatgtctaa caattcgttc aagccgacgc 5700cgcttcgcgg
cgcggcttaa ctcaagcgtt agagagctgg ggaagactat gcgcgatctg
5760ttgaaggtgg ttctaagcct cgtacttgcg atggcatttc gatcgaaagg
ggtacaaatt 5820cccactaagc gctcgggggc tgagaaagcc cagtaaggaa
acaactgtag gttcgagtcg 5880cgagatcccc cggaaccaaa ggaagtaggt
taaacccgct ccgatcaggc cgagccacgc 5940caggccgaga acattggttc
ctgtaggcat cgggattggc ggatcaaaca ctaaagctac 6000tggaacgagc
agaagtcctc cggccgccag ttgccaggcc gtaaaggtga gcagaggcac
6060gggaggttgc cacttgcggg tcagcacggt tccgaacgcc atggaaaccg
cccccgccag 6120gcccgctgcg acgccgacag gatctagcgc tgcgtttggt
gtcaacacca acagcgccac 6180gcccgcagtt ccgcaaatag cccccaggac
cgccatcaat cgtatcgggc tacctagcag 6240agcggcagag atgaacacga
ccatcagcgg ctgcacagcg cctaccgtcg ccgcgacccg 6300cccggcaggc
ggtagaccga aataaacaac aagctccaga atagcgaaat attaagtgcg
6360ccgaggatga agatgcgcat ccaccagatt cccgttggaa tctgtcggac
gatcatcacg 6420agcaataaac ccgccggcaa cgcccgcagc agcataccgg
cgacccctcg gcctcgctgt 6480tcgggctcca cgaaaacgcc ggacagatgc
gccttgtgag cgtccttggg gccgtcctcc 6540tgtttgaaga ccgacagccc
aatgatctcg ccgtcgatgt aggcgccgaa tgccacggca 6600tctcgcaacc
gttcagcgaa cgcctccatg ggctttttct cctcgtgctc gtaaacggac
6660ccgaacatct ctggagcttt cttcagggcc gacaatcgga tctcgcggaa
atcctgcacg 6720tcggccgctc caagccgtcg aatctgagcc ttaatcacaa
ttgtcaattt taatcctctg 6780tttatcggca gttcgtagag cgcgccgtgc
gcccgagcga tactgagcga agcaagtgcg 6840tcgagcagtg cccgcttgtt
cctgaaatgc cagtaaagcg ctggctgctg aacccccagc 6900cggaactgac
cccacaaggc cctagcgttt gcaatgcacc aggtcatcat tgacccaggc
6960gtgttccacc aggccgctgc ctcgcaactc ttcgcaggct tcgccgacct
gctcgcgcca 7020cttcttcacg cgggtggaat ccgatccgca catgaggcgg
aaggtttcca gcttgagcgg 7080gtacggctcc cggtgcgagc tgaaatagtc
gaacatccgt cgggccgtcg gcgacagctt 7140gcggtacttc tcccatatga
atttcgtgta gtggtcgcca gcaaacagca cgacgatttc 7200ctcgtcgatc
aggacctggc aacgggacgt tttcttgcca cggtccagga cgcggaagcg
7260gtgcagcagc gacaccgatt ccaggtgccc aacgcggtcg gacgtgaagc
ccatcgccgt 7320cgcctgtagg cgcgacaggc attcctcggc cttcgtgtaa
taccggccat tgatcgacca 7380gcccaggtcc tggcaaagct cgtagaacgt
gaaggtgatc ggctcgccga taggggtgcg 7440cttcgcgtac tccaacacct
gctgccacac cagttcgtca tcgtcggccc gcagctcgac 7500gccggtgtag
gtgatcttca cgtccttgtt gacgtggaaa atgaccttgt tttgcagcgc
7560ctcgcgcggg attttcttgt tgcgcgtggt gaacagggca gagcgggccg
tgtcgtttgg 7620catcgctcgc atcgtgtccg gccacggcgc aatatcgaac
aaggaaagct gcatttcctt 7680gatctgctgc ttcgtgtgtt tcagcaacgc
ggcctgcttg gcctcgctga cctgttttgc 7740caggtcctcg ccggcggttt
ttcgcttctt ggtcgtcata gttcctcgcg tgtcgatggt 7800catcgacttc
gccaaacctg ccgcctcctg ttcgagacga cgcgaacgct ccacggcggc
7860cgatggcgcg ggcagggcag ggggagccag ttgcacgctg tcgcgctcga
tcttggccgt 7920agcttgctgg accatcgagc cgacggactg gaaggtttcg
cggggcgcac gcatgacggt 7980gcggcttgcg atggtttcgg catcctcggc
ggaaaacccc gcgtcgatca gttcttgcct 8040gtatgccttc cggtcaaacg
tccgattcat tcaccctcct tgcgggattg ccccgactca 8100cgccggggca
atgtgccctt attcctgatt tgacccgcct ggtgccttgg tgtccagata
8160atccacctta tcggcaatga agtcggtccc gtagaccgtc tggccgtcct
tctcgtactt 8220ggtattccga atcttgccct gcacgaatac cagcgacccc
ttgcccaaat acttgccgtg 8280ggcctcggcc tgagagccaa aacacttgat
gcggaagaag tcggtgcgct cctgcttgtc 8340gccggcatcg ttgcgccact
cttcattaac cgctatatcg aaaattgctt gcggcttgtt 8400agaattgcca
tgacgtacct cggtgtcacg ggtaagatta ccgataaact ggaactgatt
8460atggcnnctc gaaattccct cggtcttgcc ttgctcgtcg gtgatgtact
tcaccagctc 8520cgcgaagtcg ctcttcttga tggagcgcat ggggacgtgc
ttggcaatca cgcgcacccc 8580ccggccgttt tagcggctaa aaaagtcatg
gctctgccct cgggcggacc acgcccatca 8640tgaccttgcc aagctcgtcc
tgcttctctt cgatcttcgc cagcagggcg aggatcgtgg 8700catcaccgaa
ccgcgccgtg cgcgggtcgt cggtgagcca gagtttcagc aggccgccca
8760ggcggcccag gtcgccattg atgcgggcca gctcgcggac gtgctcatag
tccacgacgc 8820ccgtgatttt gtagccctgg ccgacggcca gcaggtaggc
cgacaggctc atgccggccg 8880ccgccgcctt ttcctcaatc gctcttcgtt
cgtctggaag gcagtacacc ttgataggtg 8940ggctgccctt cctggttggc
ttggtttcat cagccatccg cttgccctca tctgttacgc 9000cggcggtagc
cggccagcct cgcagagcag gattcccgtt gagcaccgcc aggtgcgaat
9060aagggacagt gaagaaggaa cacccgctcg cgggtgggcc tacttcacct
atcctgcccg 9120gctgacgccg ttggatacac caaggaaagt ctacacgaac
cctttggcaa aatcctgtat 9180atcgtgcgaa aaaggatgga tataccgaaa
aaatcgctat aatgaccccg aagcagggtt 9240atgcagcgga aaagatccgt
cgaccctttc cgacgctcac cgggctggtt gccctcgccg 9300ctgggctggc
ggccgtctat ggccctgcaa acgcgccaga aacgccgtcg aagccgtgtg
9360cgagacaccg cggccgccgg cgttgtggat accacgcgga aaacttggcc
ctcactgaca 9420gatgaggggc ggacgttgac acttgagggg ccgactcacc
cggcgcggcg ttgacagatg 9480aggggcaggc tcgatttcgg ccggcgacgt
ggagctggcc agcctcgcaa atcggcgaaa 9540acgcctgatt ttacgcgagt
ttcccacaga tgatgtggac aagcctgggg ataagtgccc 9600tgcggtattg
acacttgagg ggcgcgacta ctgacagatg aggggcgcga tccttgacac
9660ttgaggggca gagtgatgac agatgagggg cgcacctatt gacatttgag
gggctgtcca 9720caggcagaaa atccagcatt tgcaagggtt tccgcccgtt
tttcggccac cgctaacctg 9780tcttttaacc tgcttttaaa ccaatattta
taaaccttgt ttttaaccag ggctgcgccc 9840tggcgcgtga ccgcgcacgc
cgaagggggg tgccccccct tctcgaaccc tcccggcccg 9900ctaacgcggg
cctcccatcc ccccaggggc tgcgcccctc ggccgcgaac ggcctcaccc
9960caaaaatggc aggccaagct agcttgcttg gtcgttccgg tacgtaccgt
gaacgtcggc 10020tcgattgtac ctgcgttcaa atactttgcg atcgtgttgc
gcgcctgccc ggtgcgtcgg 10080ctgatctcac ggatcgactg cttctctcgc
aacgccatcc gacggatgat gtttaaaagt 10140cccatgtgga tcactccgtt
gccccgtcgc tcaccgtgtt ggggggaagg tgcacatggc 10200tcagttctca
atggaaatta tctgcctaac cggctcagtt ctgcgtagaa accaacatgc
10260aagctccacc gggtgcaaag cggcagcggc ggcaggatat attcaattgt
aaatggcttc 10320atgtccggga aatctacatg gatcagcaat gagtatgatg
gtcaatatgg agaaaaagaa 10380agagtaatta ccaatttttt ttcaattcaa
aaatgtagat gtccgcagcg ttattataaa 10440atgaaagtac attttgataa
aacgacaaat tacgatccgt cgtatttata ggcgaaagca 10500ataaacaaat
tattctaatt cggaaatctt tatttcgacg tgtctacatt cacgtccaaa
10560tgggggcggc gaatt 105752510677DNAArtificial SequencepMHN vector
sequence 25ggccgcaaca gaggtggatg gacagacccg ttcttacacc ggactgggcg
cgggatagga 60tattcagatt gggatgggat tgagcttaaa gccggcgctg agaccatgct
caaggtaggc 120aatgtcctca gcgtcgagcc cggcatctat gtcgagggca
ttggtggagc gcgcttcggg 180gataccgtgc ttgtaactga gaccggatat
gaggccctca ctccgcttga tcttggcaaa 240gatatttgac gcatttatta
gtatgtgtta attttcattt gcagtgcagt attttctatt 300cgatctttat
gtaattcgtt acaattaata aatattcaaa tcagattatt gactgtcatt
360tgtatcaaat cgtgtttaat ggatattttt attataatat tgatgatatc
tcaatcaaaa 420cgtagataat aataatattt atttaatatt tttgcgtcgc
acagtgaaaa tctatatgag 480attacaaaat accgacaaca ttatttaaga
tacatagaca ttaaccctga gactgttgga 540cagagctcat tggtacctca
gatctgggta actggcctaa ctggccttgg aggagctggc 600aactcaaaat
ccctttgcca aaaaccaaca tcatgccatc caccatgctt gtatccagct
660gcgcgcaatg taccccgggc tgtgtatccc aaagcctcat gcaacctaac
agatggatcg 720tttggaaggc ctataacagc aaccacagac ttaaaacctt
gcgcctccat agacttaagc 780aaatgtgtgt acaatgtgga tcctaggccc
aacctttgat gcctatgtga cacgtaaaca 840gtactctcaa ctgtccaatc
gtaagcgttc ctagccttcc agggcccagc gtaagcaata 900ccagccacaa
caccctcaac ctcagcaacc aaccaagggt atctatcttg caacctctct
960agatcatcaa tccactcttg tggtgtttgt ggctctgtcc taaagttcac
tgtagacgtc 1020tcaatgtaat ggttaacgat atcacaaacc gcggccatat
cagctgctgt agctggccta 1080atctcaactg gtctcctctc cggagaagcc
atggtttgga tccacaaact tacaaatttc 1140tctgaagttg tatcctcagt
acttcaaaga aaatagctta caccaatttt ttcttgtttt 1200cacaaatgcc
gaacttggtt ccttatatag gaaaactcaa gggcaaaaat gacacggaaa
1260aatataaaag gataagtagt gggggataag attcctttgt gataaggtta
ctttccgccc 1320ttacattttc caccttacat gtgtcctcta tgtctctttc
acaatcaccg accttatctt 1380cttcttttca ttgttgtcgt cagtgcttac
gtcttcaaga ttcttttctt cgcctggttc 1440ttctttttca atttctacgt
attcttcttc gtattctggc agtataggat cttgtatctg 1500tacattcttc
atttttgaac ataggttgca tatgtgccgc atattgatct gcttcttgct
1560gagctcacat aatacttcca tagtttttcc cgtaaacatt ggattcttga
tgctacatct 1620tggataatta ccttctggcc ggccgcgaat tcgcttcaag
acgtgctcaa atcactattt 1680ccacacccct atatttctat tgcactccct
tttaactgtt ttttattaca aaaatgccct 1740ggaaaatgca ctcccttttt
gtgtttgtta tttagtgaaa cgatgttgtc aggtaattta 1800tttgtcagtc
tactatggtg gcccattata ttaatagcaa ctgtcggtcc aatagacgac
1860gtcgattttc tgcatttgtt taaccacgtg gattttatga cattttatat
tagttaattt 1920gtaaaaccta cccaattaaa gacctcatat gttctaaaga
ctaatactta atgataacaa 1980ttttctttta gtgaagaaag ggataattag
taaatatgga acaagggcag aagatttatt 2040aaagccggta agagacaaca
acgtaggtac gtggagtgtc ttaggtgact tacccacata 2100acataaagtg
acattaacaa acatagctaa tgctcctatt tgaatagtgc atatcagcat
2160accttattac atatagatag gagcaaactc tagctagatt gttgagagag
ctcggtaccg 2220gatccgaaga cttaacctga tcttgcttca cgtacaccat
catctttcag aatctccacc 2280aaaagtggaa caattctgaa ttccccaaag
agagtgttag aaatctcagc tatgctcaga 2340caataggtct tgttcgtctt
tacaactttg aaacatgtgg aggtagtgta tgctgcctta 2400gtagaactag
aggaaactct ggttatcctt gatctggatg tagaatcaaa cacagcagag
2460gcaggattca acctagcttg aacaccatct aacattgttc caaacacccc
tctcaaggta 2520tgattacggt agaagatcaa agggtaagga tcagtgtaaa
ctccagtcac acacgaattc 2580ggacatctag ctgaagcttg gcaaggaatc
gatccaggtc ttgtaaaggc attgaaagta 2640tatggtgaat gtaatgtagc
tgtcttgttg ctcacagtca ttggatacag taacgctggg 2700ctaaagtagg
aacttccacg ttgatagaga aagtggctag taccaacagt aagaatcctt
2760ccctcagctc ccatcaatgt tacagtgttt ggtggaacag taaggactgg
atcttctccc 2820aatgatgtgc taaccttgat actgagaata gcttgttgta
tcctcttacc tccaaatctt 2880cctggtttgt atgatgactt agccattcga
atctgatagt cttgctcatc aggacaagta 2940tcattgtacc tcttgtagat
aacatacttt ccctcttgca cagtatcact aggactgtta 3000ggtttcaaac
caccatagac agagaaccag actctggagt caatgaagct accacctcca
3060actcctggat agttagctac ccaatcccca aacaatgtag tgacatccaa
atctttctca 3120tggtattgac catcaaaacc caatctgcca tgcaccatcc
ttgtagggac tgcagaattg 3180tagtcctctt cttcagtttc tgttacttta
ctgcatagca tatcacagcc caaaggtgta 3240gcagatacag agcaactttt
gcgattctgt gtatcatcaa gattgatact gcgaagagtt 3300gagaagaaca
ctctaccagt agcagatgta cgaagaactc caagtgctaa gtactgataa
3360gagtgagaat ggtcacgaca gccagataga atgacattgt gtgtatagca
atagtgtgta 3420gcactcatgt caaatgaggg tatccgagtg cacccactcc
cagttgtggg tgcaggaatg 3480aagttcagat gttcctggaa agctgaagga
tagaaagatg taacatctga agcatcatct 3540acaatgagtt ctttgccaat
acctccaatg tagtctggat catgaattgg ggctccccaa 3600cctgaattgt
tggcagcccc attgatctga taggataggc tggtgattgc attcatgatg
3660gtagtttctg tgttgagtaa tgcaagtgga ctttcaagtg caacttgttt
gtagattcgg 3720tccacaacat cctggttgga gcctagggta ctggtaatct
tctcttcagc cctagagatt 3780cgtgtgggta tgccaactaa gtctgagggg
gtgcttgctc ccatagaata gagtaatgat 3840gcaacagaga ttgctagggt
gacaacagta agaaagagaa tggctatccg aaagataagc 3900ctccaagtgt
tcttggcttc cctctcatca ttctcaagag ccacttgtga aactgctcgg
3960tccatggttt ggatccgcga tttggtgtat cgagattggt tatgaaattc
agatgctagt 4020gtaatgtatt ggtaatttgg gaagatataa taggaagcaa
ggctatttat ccatttctga 4080aaaggcgaaa tggcgtcacc gcgagcgtca
cgctctagtc gaccatgtac gtaagcgctt 4140acgtttttgg tggaccccct
cgaccatgta cgtaagcgct tacgtttttg gtggaccccc 4200tcgaccatgt
acgtaagcgc ttacgttttt ggtggacccc ctcgaccatg tacgtaagcg
4260cttacgtttt tggtggaccc cctcgacgga tcccccctcg accctagacg
tatctattca 4320aaagtcgtta atggctgcgg atcaagaaaa agttggaata
gaaacagaat acccgcgaaa 4380ttcaggcccg gttgccatgt cctacacgcc
gaaataaacg accaaattag tagaaaaata 4440aaaactagct cagatactta
cgtcacgtct tgcgcactga tttgaaaaat ctcaatataa 4500acaaagacgg
ccacaagaaa aaaccaaaac accgatattc attaatctta tctagtttct
4560caaaaaaatt catatcttcc acaccctcga gatctagata aacttaatta
agcggccgat 4620ccggtgagta atattgtacg gctaagagcg aatttggcct
gtagacctca attgcgagct 4680ttctaatttc aaactattcg ggcctaactt
ttggtgtgat gatgctgact ggcaggatat 4740ataccgttgt aatttgagct
cgtgtgaata agtcgctgtg tatgtttgtt tgattgtttc 4800tgttggagtg
cagcccattt caccggacaa gtcggctaga ttgatttagc cctgatgaac
4860tgccgagggg aagccatctt gagcgcggaa tgggaatgga tcgaaccggg
agcacaggat 4920gacgcctaac aattcattca agccgacacc gcttcgcggc
gcggcttaat tcaggagtta 4980aacatcatga gggaagcggt gatcgccgaa
gtatcgactc aactatcaga ggtagttggc 5040gtcatcgagc gccatctcga
accgacgttg ctggccgtac atttgtacgg ctccgcagtg 5100gatggcggcc
tgaagccaca cagtgatatt gatttgctgg ttacggtgac cgtaaggctt
5160gatgaaacaa cgcggcgagc tttgatcaac gaccttttgg aaacttcggc
ttcccctgga 5220gagagcgaga ttctccgcgc tgtagaagtc accattgttg
tgcacgacga catcattccg 5280tggcgttatc cagctaagcg cgaactgcaa
tttggagaat ggcagcgcaa tgacattctt 5340gcaggtatct tcgagccagc
cacgatcgac attgatctgg ctatcttgct gacaaaagca 5400agagaacata
gcgttgcctt ggtaggtcca gcggcggagg aactctttga tccggttcct
5460gaacaggatc tatttgaggc gctaaatgaa accttaacgc tatggaactc
gccgcccgac 5520tgggctggcg atgagcgaaa tgtagtgctt acgttgtccc
gcatttggta cagcgcagta 5580accggcaaaa tcgcgccgaa ggatgtcgct
gccgactggg caatggagcg cctgccggcc 5640cagtatcagc ccgtcatact
tgaagctagg caggcttatc ttggacaaga agatcgcttg 5700gcctcgcgcg
cagatcagtt ggaagaattt gttcactacg tgaaaggcga gatcaccaag
5760gtagtcggca aataatgtct aacaattcgt tcaagccgac gccgcttcgc
ggcgcggctt 5820aactcaagcg ttagagagct ggggaagact atgcgcgatc
tgttgaaggt ggttctaagc 5880ctcgtacttg cgatggcatt tcgatcgaaa
ggggtacaaa ttcccactaa gcgctcgggg 5940gctgagaaag cccagtaagg
aaacaactgt aggttcgagt cgcgagatcc cccggaacca 6000aaggaagtag
gttaaacccg ctccgatcag gccgagccac gccaggccga gaacattggt
6060tcctgtaggc atcgggattg gcggatcaaa cactaaagct actggaacga
gcagaagtcc 6120tccggccgcc agttgccagg ccgtaaaggt gagcagaggc
acgggaggtt gccacttgcg 6180ggtcagcacg gttccgaacg ccatggaaac
cgcccccgcc aggcccgctg cgacgccgac 6240aggatctagc gctgcgtttg
gtgtcaacac caacagcgcc acgcccgcag ttccgcaaat 6300agcccccagg
accgccatca atcgtatcgg gctacctagc agagcggcag agatgaacac
6360gaccatcagc ggctgcacag cgcctaccgt cgccgcgacc cgcccggcag
gcggtagacc 6420gaaataaaca acaagctcca gaatagcgaa atattaagtg
cgccgaggat gaagatgcgc 6480atccaccaga ttcccgttgg aatctgtcgg
acgatcatca cgagcaataa acccgccggc 6540aacgcccgca gcagcatacc
ggcgacccct cggcctcgct gttcgggctc cacgaaaacg 6600ccggacagat
gcgccttgtg agcgtccttg gggccgtcct cctgtttgaa gaccgacagc
6660ccaatgatct cgccgtcgat gtaggcgccg aatgccacgg catctcgcaa
ccgttcagcg 6720aacgcctcca tgggcttttt ctcctcgtgc tcgtaaacgg
acccgaacat ctctggagct 6780ttcttcaggg ccgacaatcg gatctcgcgg
aaatcctgca cgtcggccgc tccaagccgt 6840cgaatctgag ccttaatcac
aattgtcaat tttaatcctc tgtttatcgg cagttcgtag 6900agcgcgccgt
gcgcccgagc gatactgagc gaagcaagtg cgtcgagcag tgcccgcttg
6960ttcctgaaat gccagtaaag cgctggctgc tgaaccccca gccggaactg
accccacaag 7020gccctagcgt ttgcaatgca ccaggtcatc attgacccag
gcgtgttcca ccaggccgct 7080gcctcgcaac tcttcgcagg cttcgccgac
ctgctcgcgc cacttcttca cgcgggtgga 7140atccgatccg cacatgaggc
ggaaggtttc cagcttgagc gggtacggct cccggtgcga 7200gctgaaatag
tcgaacatcc gtcgggccgt cggcgacagc ttgcggtact tctcccatat
7260gaatttcgtg tagtggtcgc cagcaaacag cacgacgatt tcctcgtcga
tcaggacctg 7320gcaacgggac gttttcttgc cacggtccag gacgcggaag
cggtgcagca gcgacaccga 7380ttccaggtgc ccaacgcggt cggacgtgaa
gcccatcgcc gtcgcctgta ggcgcgacag 7440gcattcctcg gccttcgtgt
aataccggcc attgatcgac cagcccaggt cctggcaaag 7500ctcgtagaac
gtgaaggtga tcggctcgcc gataggggtg cgcttcgcgt actccaacac
7560ctgctgccac accagttcgt catcgtcggc ccgcagctcg acgccggtgt
aggtgatctt 7620cacgtccttg ttgacgtgga aaatgacctt gttttgcagc
gcctcgcgcg ggattttctt 7680gttgcgcgtg gtgaacaggg cagagcgggc
cgtgtcgttt ggcatcgctc gcatcgtgtc 7740cggccacggc gcaatatcga
acaaggaaag ctgcatttcc ttgatctgct gcttcgtgtg 7800tttcagcaac
gcggcctgct tggcctcgct gacctgtttt gccaggtcct cgccggcggt
7860ttttcgcttc ttggtcgtca tagttcctcg cgtgtcgatg gtcatcgact
tcgccaaacc 7920tgccgcctcc tgttcgagac gacgcgaacg ctccacggcg
gccgatggcg cgggcagggc 7980agggggagcc agttgcacgc tgtcgcgctc
gatcttggcc gtagcttgct ggaccatcga 8040gccgacggac tggaaggttt
cgcggggcgc acgcatgacg gtgcggcttg cgatggtttc 8100ggcatcctcg
gcggaaaacc ccgcgtcgat cagttcttgc ctgtatgcct tccggtcaaa
8160cgtccgattc attcaccctc cttgcgggat tgccccgact cacgccgggg
caatgtgccc 8220ttattcctga tttgacccgc ctggtgcctt ggtgtccaga
taatccacct tatcggcaat 8280gaagtcggtc ccgtagaccg tctggccgtc
cttctcgtac ttggtattcc gaatcttgcc 8340ctgcacgaat accagcgacc
ccttgcccaa atacttgccg tgggcctcgg cctgagagcc 8400aaaacacttg
atgcggaaga agtcggtgcg ctcctgcttg tcgccggcat cgttgcgcca
8460ctcttcatta accgctatat cgaaaattgc ttgcggcttg ttagaattgc
catgacgtac 8520ctcggtgtca cgggtaagat taccgataaa ctggaactga
ttatggcnnc tcgaaattcc 8580ctcggtcttg ccttgctcgt cggtgatgta
cttcaccagc tccgcgaagt cgctcttctt 8640gatggagcgc atggggacgt
gcttggcaat cacgcgcacc ccccggccgt tttagcggct 8700aaaaaagtca
tggctctgcc ctcgggcgga ccacgcccat catgaccttg ccaagctcgt
8760cctgcttctc ttcgatcttc gccagcaggg cgaggatcgt ggcatcaccg
aaccgcgccg 8820tgcgcgggtc gtcggtgagc cagagtttca gcaggccgcc
caggcggccc aggtcgccat 8880tgatgcgggc cagctcgcgg acgtgctcat
agtccacgac gcccgtgatt ttgtagccct 8940ggccgacggc cagcaggtag
gccgacaggc tcatgccggc cgccgccgcc ttttcctcaa 9000tcgctcttcg
ttcgtctgga aggcagtaca ccttgatagg tgggctgccc ttcctggttg
9060gcttggtttc atcagccatc cgcttgccct catctgttac gccggcggta
gccggccagc 9120ctcgcagagc aggattcccg ttgagcaccg ccaggtgcga
ataagggaca gtgaagaagg 9180aacacccgct cgcgggtggg cctacttcac
ctatcctgcc cggctgacgc cgttggatac 9240accaaggaaa gtctacacga
accctttggc aaaatcctgt atatcgtgcg aaaaaggatg 9300gatataccga
aaaaatcgct ataatgaccc cgaagcaggg ttatgcagcg gaaaagatcc
9360gtcgaccctt tccgacgctc accgggctgg ttgccctcgc cgctgggctg
gcggccgtct 9420atggccctgc aaacgcgcca gaaacgccgt cgaagccgtg
tgcgagacac cgcggccgcc 9480ggcgttgtgg ataccacgcg gaaaacttgg
ccctcactga cagatgaggg gcggacgttg 9540acacttgagg ggccgactca
cccggcgcgg cgttgacaga tgaggggcag gctcgatttc 9600ggccggcgac
gtggagctgg ccagcctcgc aaatcggcga aaacgcctga ttttacgcga
9660gtttcccaca gatgatgtgg acaagcctgg ggataagtgc cctgcggtat
tgacacttga 9720ggggcgcgac tactgacaga tgaggggcgc gatccttgac
acttgagggg cagagtgatg 9780acagatgagg
ggcgcaccta ttgacatttg aggggctgtc cacaggcaga aaatccagca
9840tttgcaaggg tttccgcccg tttttcggcc accgctaacc tgtcttttaa
cctgctttta 9900aaccaatatt tataaacctt gtttttaacc agggctgcgc
cctggcgcgt gaccgcgcac 9960gccgaagggg ggtgcccccc cttctcgaac
cctcccggcc cgctaacgcg ggcctcccat 10020ccccccaggg gctgcgcccc
tcggccgcga acggcctcac cccaaaaatg gcaggccaag 10080ctagcttgct
tggtcgttcc ggtacgtacc gtgaacgtcg gctcgattgt acctgcgttc
10140aaatactttg cgatcgtgtt gcgcgcctgc ccggtgcgtc ggctgatctc
acggatcgac 10200tgcttctctc gcaacgccat ccgacggatg atgtttaaaa
gtcccatgtg gatcactccg 10260ttgccccgtc gctcaccgtg ttggggggaa
ggtgcacatg gctcagttct caatggaaat 10320tatctgccta accggctcag
ttctgcgtag aaaccaacat gcaagctcca ccgggtgcaa 10380agcggcagcg
gcggcaggat atattcaatt gtaaatggct tcatgtccgg gaaatctaca
10440tggatcagca atgagtatga tggtcaatat ggagaaaaag aaagagtaat
taccaatttt 10500ttttcaattc aaaaatgtag atgtccgcag cgttattata
aaatgaaagt acattttgat 10560aaaacgacaa attacgatcc gtcgtattta
taggcgaaag caataaacaa attattctaa 10620ttcggaaatc tttatttcga
cgtgtctaca ttcacgtcca aatgggggcg gcgaatt 106772610603DNAArtificial
SequencepCHN vector sequence 26tgcgtagaaa ccaacatgca agctccaccg
ggtgcaaagc ggcagcggcg gcaggatata 60ttcaattgta aatggcttca tgtccgggaa
atctacatgg atcagcaatg agtatgatgg 120tcaatatgga gaaaaagaaa
gagtaattac caattttttt tcaattcaaa aatgtagatg 180tccgcagcgt
tattataaaa tgaaagtaca ttttgataaa acgacaaatt acgatccgtc
240gtatttatag gcgaaagcaa taaacaaatt attctaattc ggaaatcttt
atttcgacgt 300gtctacattc acgtccaaat gggggcggcg aattggccgc
aacagaggtg gatggacaga 360cccgttctta caccggactg ggcgcgggat
aggatattca gattgggatg ggattgagct 420taaagccggc gctgagacca
tgctcaaggt aggcaatgtc ctcagcgtcg agcccggcat 480ctatgtcgag
ggcattggtg gagcgcgctt cggggatacc gtgcttgtaa ctgagaccgg
540atatgaggcc ctcactccgc ttgatcttgg caaagatatt tgacgcattt
attagtatgt 600gttaattttc atttgcagtg cagtattttc tattcgatct
ttatgtaatt cgttacaatt 660aataaatatt caaatcagat tattgactgt
catttgtatc aaatcgtgtt taatggatat 720ttttattata atattgatga
tatctcaatc aaaacgtaga taataataat atttatttaa 780tatttttgcg
tcgcacagtg aaaatctata tgagattaca aaataccgac aacattattt
840aagatacata gacattaacc ctgagactgt tggacagagc tcattggtac
ctcagatctg 900ggtaactggc ctaactggcc ttggaggagc tggcaactca
aaatcccttt gccaaaaacc 960aacatcatgc catccaccat gcttgtatcc
agctgcgcgc aatgtacccc gggctgtgta 1020tcccaaagcc tcatgcaacc
taacagatgg atcgtttgga aggcctataa cagcaaccac 1080agacttaaaa
ccttgcgcct ccatagactt aagcaaatgt gtgtacaatg tggatcctag
1140gcccaacctt tgatgcctat gtgacacgta aacagtactc tcaactgtcc
aatcgtaagc 1200gttcctagcc ttccagggcc cagcgtaagc aataccagcc
acaacaccct caacctcagc 1260aaccaaccaa gggtatctat cttgcaacct
ctctagatca tcaatccact cttgtggtgt 1320ttgtggctct gtcctaaagt
tcactgtaga cgtctcaatg taatggttaa cgatatcaca 1380aaccgcggcc
atatcagctg ctgtagctgg cctaatctca actggtctcc tctccggaga
1440agccatggtt tggatccaca aacttacaaa tttctctgaa gttgtatcct
cagtacttca 1500aagaaaatag cttacaccaa attttttctt gttttcacaa
atgccgaact tggttcctta 1560tataggaaaa ctcaagggca aaaatgacac
ggaaaaatat aaaaggataa gtagtggggg 1620ataagattcc tttgtgataa
ggttactttc cgcccttaca ttttccacct tacatgtgtc 1680ctctatgtct
ctttcacaat caccgacctt atcttcttct tttcattgtt gtcgtcagtg
1740cttacgtctt caagattctt ttcttcgcct ggttcttctt tttcaatttc
tacgtattct 1800tcttcgtatt ctggcagtat aggatcttgt atctgtacat
tcttcatttt tgaacatagg 1860ttgcatatgt gccgcatatt gatctgcttc
ttgctgagct cacataatac ttccatagtt 1920tttcccgtaa acattggatt
cttgatgcta catcttggat aattaccttc tggccggccg 1980cgaattcgct
tcaagacgtg ctcaaatcac tatttccaca cccctatatt tctattgcac
2040tcccttttaa ctgtttttta ttacaaaaat gccctggaaa atgcactccc
tttttgtgtt 2100tgttatttag tgaaacgatg ttgtcaggta atttatttgt
cagtctacta tggtggccca 2160ttatattaat agcaactgtc ggtccaatag
acgacgtcga ttttctgcat ttgtttaacc 2220acgtggattt tatgacattt
tatattagtt aatttgtaaa acctacccaa ttaaagacct 2280catatgttct
aaagactaat acttaatgat aacaattttc ttttagtgaa gaaagggata
2340attagtaaat atggaacaag ggcagaagat ttattaaagc cggtaagaga
caacaacgta 2400ggtacgtgga gtgtcttagg tgacttaccc acataacata
aagtgacatt aacaaacata 2460gctaatgctc ctatttgaat agtgcatatc
agcatacctt attacatata gataggagca 2520aactctagct agattgttga
gagagctcgg taccggatcc gaagacttaa cctgatcttg 2580cttcacgtac
accatcatct ttcagaatct ccaccaaaag tggaacaatt ctgaattccc
2640caaagagagt gttagaaatc tcagctatgc tcagacaata ggtcttgttc
gtctttacaa 2700ctttgaaaca tgtggaggta gtgtatgctg ccttagtaga
actagaggaa actctggtta 2760tccttgatct ggatgtagaa tcaaacacag
cagaggcagg attcaaccta gcttgaacac 2820catctaacat tgttccaaac
acccctctca aggtatgatt acggtagaag atcaaagggt 2880aaggatcagt
gtaaactcca gtcacacacg aattcggaca tctagctgaa gcttggcaag
2940gaatcgatcc aggtcttgta aaggcattga aagtatatgg tgaatgtaat
gtagctgtct 3000tgttgctcac agtcattgga tacagtaacg ctgggctaaa
gtaggaactt ccacgttgat 3060agagaaagtg gctagtacca acagtaagaa
tccttccctc agctcccatc aatgttacag 3120tgtttggtgg aacagtaagg
actggatctt ctcccaatga tgtgctaacc ttgatactga 3180gaatagcttg
ttgtatcctc ttacctccaa atcttcctgg tttgtatgat gacttagcca
3240ttcgaatctg atagtcttgc tcatcaggac aagtatcatt gtacctcttg
tagataacat 3300actttccctc ttgcacagta tcactaggac tgttaggttt
caaaccacca tagacagaga 3360accagactct ggagtcaatg aagctaccac
ctccaactcc tggatagtta gctacccaat 3420ccccaaacaa tgtagtgaca
tccaaatctt tctcatggta ttgaccatca aaacccaatc 3480tgccatgcac
catccttgta gggactgcag aattgtagtc ctcttcttca gtttctgtta
3540ctttactgca tagcatatca cagcccaaag gtgtagcaga tacagagcaa
cttttgcgat 3600tctgtgtatc atcaagattg atactgcgaa gagttgagaa
gaacactcta ccagtagcag 3660atgtacgaag aactccaagt gctaagtact
gataagagtg agaatggtca cgacagccag 3720atagaatgac attgtgtgta
tagcaatagt gtgtagcact catgtcaaat gagggtatcc 3780gagtgcaccc
actcccagtt gtgggtgcag gaatgaagtt cagatgttcc tggaaagctg
3840aaggatagaa agatgtaaca tctgaagcat catctacaat gagttctttg
ccaatacctc 3900caatgtagtc tggatcatga attggggctc cccaacctga
attgttggca gccccattga 3960tctgatagga taggctggtg attgcattca
tgatggtagt ttctgtgttg agtaatgcaa 4020gtggactttc aagtgcaact
tgtttgtaga ttcggtccac aacatcctgg ttggagccta 4080gggtactggt
aatcttctct tcagccctag agattcgtgt gggtatgcca actaagtctg
4140agggggtgct tgctcccata gaatagagta atgatgcaac agagattgct
agggtgacaa 4200cagtaagaaa gagaatggct atccgaaaga taagcctcca
agtgttcttg gcttccctct 4260catcattctc aagagccact tgtgaaactg
ctcggtccat ggtttggatc cacaaactta 4320caaatttctc tgaagttgta
tcctcagtac ttcaaagaaa atagcttaca ccaaattttt 4380tcttgttttc
acaaatgccg aacttggttc cttatatagg aaaactcaag ggcaaaaatg
4440acacggaaaa atataaaagg ataagtagtg ggggataaga ttcctttgtg
ataaggttac 4500tttccgccct tacattttcc accttacatg tgtcctctat
gtctctttca caatcaccga 4560ccttatcttc ttcttttcat tgttgtcgtc
agtgcttacg tcttcaagat tcttttcttc 4620gcctggttct tctttttcaa
tttctacgta ttcttcttcg tattctggca gtataggatc 4680ttgtatctgt
acattcttca tttttgaaca taggttgcat atgtgccgca tattgatctg
4740cttcttgctg agctcacata atacttccat agtttttccc gtaaacattg
gattcttgat 4800gctacatctt ggataattac cttctggcgc gcctttgccc
gggctttcct gcagggttta 4860aacttaatta agcggccgat ccggtgagta
atattgtacg gctaagagcg aatttggcct 4920gtagacctca attgcgagct
ttctaatttc aaactattcg ggcctaactt ttggtgtgat 4980gatgctgact
ggcaggatat ataccgttgt aatttgagct cgtgtgaata agtcgctgtg
5040tatgtttgtt tgattgtttc tgttggagtg cagcccattt caccggacaa
gtcggctaga 5100ttgatttagc cctgatgaac tgccgagggg aagccatctt
gagcgcggaa tgggaatgga 5160tcgaaccggg agcacaggat gacgcctaac
aattcattca agccgacacc gcttcgcggc 5220gcggcttaat tcaggagtta
aacatcatga gggaagcggt gatcgccgaa gtatcgactc 5280aactatcaga
ggtagttggc gtcatcgagc gccatctcga accgacgttg ctggccgtac
5340atttgtacgg ctccgcagtg gatggcggcc tgaagccaca cagtgatatt
gatttgctgg 5400ttacggtgac cgtaaggctt gatgaaacaa cgcggcgagc
tttgatcaac gaccttttgg 5460aaacttcggc ttcccctgga gagagcgaga
ttctccgcgc tgtagaagtc accattgttg 5520tgcacgacga catcattccg
tggcgttatc cagctaagcg cgaactgcaa tttggagaat 5580ggcagcgcaa
tgacattctt gcaggtatct tcgagccagc cacgatcgac attgatctgg
5640ctatcttgct gacaaaagca agagaacata gcgttgcctt ggtaggtcca
gcggcggagg 5700aactctttga tccggttcct gaacaggatc tatttgaggc
gctaaatgaa accttaacgc 5760tatggaactc gccgcccgac tgggctggcg
atgagcgaaa tgtagtgctt acgttgtccc 5820gcatttggta cagcgcagta
accggcaaaa tcgcgccgaa ggatgtcgct gccgactggg 5880caatggagcg
cctgccggcc cagtatcagc ccgtcatact tgaagctagg caggcttatc
5940ttggacaaga agatcgcttg gcctcgcgcg cagatcagtt ggaagaattt
gttcactacg 6000tgaaaggcga gatcaccaag gtagtcggca aataatgtct
aacaattcgt tcaagccgac 6060gccgcttcgc ggcgcggctt aactcaagcg
ttagagagct ggggaagact atgcgcgatc 6120tgttgaaggt ggttctaagc
ctcgtacttg cgatggcatt tcgatcgaaa ggggtacaaa 6180ttcccactaa
gcgctcgggg gctgagaaag cccagtaagg aaacaactgt aggttcgagt
6240cgcgagatcc cccggaacca aaggaagtag gttaaacccg ctccgatcag
gccgagccac 6300gccaggccga gaacattggt tcctgtaggc atcgggattg
gcggatcaaa cactaaagct 6360actggaacga gcagaagtcc tccggccgcc
agttgccagg ccgtaaaggt gagcagaggc 6420acgggaggtt gccacttgcg
ggtcagcacg gttccgaacg ccatggaaac cgcccccgcc 6480aggcccgctg
cgacgccgac aggatctagc gctgcgtttg gtgtcaacac caacagcgcc
6540acgcccgcag ttccgcaaat agcccccagg accgccatca atcgtatcgg
gctacctagc 6600agagcggcag agatgaacac gaccatcagc ggctgcacag
cgcctaccgt cgccgcgacc 6660cgcccggcag gcggtagacc gaaataaaca
acaagctcca gaatagcgaa atattaagtg 6720cgccgaggat gaagatgcgc
atccaccaga ttcccgttgg aatctgtcgg acgatcatca 6780cgagcaataa
acccgccggc aacgcccgca gcagcatacc ggcgacccct cggcctcgct
6840gttcgggctc cacgaaaacg ccggacagat gcgccttgtg agcgtccttg
gggccgtcct 6900cctgtttgaa gaccgacagc ccaatgatct cgccgtcgat
gtaggcgccg aatgccacgg 6960catctcgcaa ccgttcagcg aacgcctcca
tgggcttttt ctcctcgtgc tcgtaaacgg 7020acccgaacat ctctggagct
ttcttcaggg ccgacaatcg gatctcgcgg aaatcctgca 7080cgtcggccgc
tccaagccgt cgaatctgag ccttaatcac aattgtcaat tttaatcctc
7140tgtttatcgg cagttcgtag agcgcgccgt gcgcccgagc gatactgagc
gaagcaagtg 7200cgtcgagcag tgcccgcttg ttcctgaaat gccagtaaag
cgctggctgc tgaaccccca 7260gccggaactg accccacaag gccctagcgt
ttgcaatgca ccaggtcatc attgacccag 7320gcgtgttcca ccaggccgct
gcctcgcaac tcttcgcagg cttcgccgac ctgctcgcgc 7380cacttcttca
cgcgggtgga atccgatccg cacatgaggc ggaaggtttc cagcttgagc
7440gggtacggct cccggtgcga gctgaaatag tcgaacatcc gtcgggccgt
cggcgacagc 7500ttgcggtact tctcccatat gaatttcgtg tagtggtcgc
cagcaaacag cacgacgatt 7560tcctcgtcga tcaggacctg gcaacgggac
gttttcttgc cacggtccag gacgcggaag 7620cggtgcagca gcgacaccga
ttccaggtgc ccaacgcggt cggacgtgaa gcccatcgcc 7680gtcgcctgta
ggcgcgacag gcattcctcg gccttcgtgt aataccggcc attgatcgac
7740cagcccaggt cctggcaaag ctcgtagaac gtgaaggtga tcggctcgcc
gataggggtg 7800cgcttcgcgt actccaacac ctgctgccac accagttcgt
catcgtcggc ccgcagctcg 7860acgccggtgt aggtgatctt cacgtccttg
ttgacgtgga aaatgacctt gttttgcagc 7920gcctcgcgcg ggattttctt
gttgcgcgtg gtgaacaggg cagagcgggc cgtgtcgttt 7980ggcatcgctc
gcatcgtgtc cggccacggc gcaatatcga acaaggaaag ctgcatttcc
8040ttgatctgct gcttcgtgtg tttcagcaac gcggcctgct tggcctcgct
gacctgtttt 8100gccaggtcct cgccggcggt ttttcgcttc ttggtcgtca
tagttcctcg cgtgtcgatg 8160gtcatcgact tcgccaaacc tgccgcctcc
tgttcgagac gacgcgaacg ctccacggcg 8220gccgatggcg cgggcagggc
agggggagcc agttgcacgc tgtcgcgctc gatcttggcc 8280gtagcttgct
ggaccatcga gccgacggac tggaaggttt cgcggggcgc acgcatgacg
8340gtgcggcttg cgatggtttc ggcatcctcg gcggaaaacc ccgcgtcgat
cagttcttgc 8400ctgtatgcct tccggtcaaa cgtccgattc attcaccctc
cttgcgggat tgccccgact 8460cacgccgggg caatgtgccc ttattcctga
tttgacccgc ctggtgcctt ggtgtccaga 8520taatccacct tatcggcaat
gaagtcggtc ccgtagaccg tctggccgtc cttctcgtac 8580ttggtattcc
gaatcttgcc ctgcacgaat accagcgacc ccttgcccaa atacttgccg
8640tgggcctcgg cctgagagcc aaaacacttg atgcggaaga agtcggtgcg
ctcctgcttg 8700tcgccggcat cgttgcgcca ctcttcatta accgctatat
cgaaaattgc ttgcggcttg 8760ttagaattgc catgacgtac ctcggtgtca
cgggtaagat taccgataaa ctggaactga 8820ttatggcnnc tcgaaattcc
ctcggtcttg ccttgctcgt cggtgatgta cttcaccagc 8880tccgcgaagt
cgctcttctt gatggagcgc atggggacgt gcttggcaat cacgcgcacc
8940ccccggccgt tttagcggct aaaaaagtca tggctctgcc ctcgggcgga
ccacgcccat 9000catgaccttg ccaagctcgt cctgcttctc ttcgatcttc
gccagcaggg cgaggatcgt 9060ggcatcaccg aaccgcgccg tgcgcgggtc
gtcggtgagc cagagtttca gcaggccgcc 9120caggcggccc aggtcgccat
tgatgcgggc cagctcgcgg acgtgctcat agtccacgac 9180gcccgtgatt
ttgtagccct ggccgacggc cagcaggtag gccgacaggc tcatgccggc
9240cgccgccgcc ttttcctcaa tcgctcttcg ttcgtctgga aggcagtaca
ccttgatagg 9300tgggctgccc ttcctggttg gcttggtttc atcagccatc
cgcttgccct catctgttac 9360gccggcggta gccggccagc ctcgcagagc
aggattcccg ttgagcaccg ccaggtgcga 9420ataagggaca gtgaagaagg
aacacccgct cgcgggtggg cctacttcac ctatcctgcc 9480cggctgacgc
cgttggatac accaaggaaa gtctacacga accctttggc aaaatcctgt
9540atatcgtgcg aaaaaggatg gatataccga aaaaatcgct ataatgaccc
cgaagcaggg 9600ttatgcagcg gaaaagatcc gtcgaccctt tccgacgctc
accgggctgg ttgccctcgc 9660cgctgggctg gcggccgtct atggccctgc
aaacgcgcca gaaacgccgt cgaagccgtg 9720tgcgagacac cgcggccgcc
ggcgttgtgg ataccacgcg gaaaacttgg ccctcactga 9780cagatgaggg
gcggacgttg acacttgagg ggccgactca cccggcgcgg cgttgacaga
9840tgaggggcag gctcgatttc ggccggcgac gtggagctgg ccagcctcgc
aaatcggcga 9900aaacgcctga ttttacgcga gtttcccaca gatgatgtgg
acaagcctgg ggataagtgc 9960cctgcggtat tgacacttga ggggcgcgac
tactgacaga tgaggggcgc gatccttgac 10020acttgagggg cagagtgatg
acagatgagg ggcgcaccta ttgacatttg aggggctgtc 10080cacaggcaga
aaatccagca tttgcaaggg tttccgcccg tttttcggcc accgctaacc
10140tgtcttttaa cctgctttta aaccaatatt tataaacctt gtttttaacc
agggctgcgc 10200cctggcgcgt gaccgcgcac gccgaagggg ggtgcccccc
cttctcgaac cctcccggcc 10260cgctaacgcg ggcctcccat ccccccaggg
gctgcgcccc tcggccgcga acggcctcac 10320cccaaaaatg gcaggccaag
ctagcttgct tggtcgttcc ggtacgtacc gtgaacgtcg 10380gctcgattgt
acctgcgttc aaatactttg cgatcgtgtt gcgcgcctgc ccggtgcgtc
10440ggctgatctc acggatcgac tgcttctctc gcaacgccat ccgacggatg
atgtttaaaa 10500gtcccatgtg gatcactccg ttgccccgtc gctcaccgtg
ttggggggaa ggtgcacatg 10560gctcagttct caatggaaat tatctgccta
accggctcag ttc 10603
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References