Production of human interferon alfa 2b in plants of Nicotiana excelsior by Agrobacterium-mediated transient expression
Human interferon α2b gene was transiently expressed in Nicotiana excelsior plants. Fusion with N. plumbaginifolia calreticulin signal peptide for improved apoplast targeting and carrying out the expression under optimized conditions resulted in maximal interferon activity of 3.2 • 10³ IU/g fresh wei...
Gespeichert in:
| Datum: | 2010 |
|---|---|
| Hauptverfasser: | , , , , , |
| Format: | Artikel |
| Sprache: | English |
| Veröffentlicht: |
Інститут клітинної біології та генетичної інженерії НАН України
2010
|
| Schriftenreihe: | Цитология и генетика |
| Schlagworte: | |
| Online Zugang: | https://nasplib.isofts.kiev.ua/handle/123456789/66797 |
| Tags: |
Tag hinzufügen
Keine Tags, Fügen Sie den ersten Tag hinzu!
|
| Назва журналу: | Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| Zitieren: | Production of human interferon alfa 2b in plants of Nicotiana excelsior by Agrobacterium-mediated transient expression / Y.R. Sindarovska, I.M. Gerasymenko, Y.V. Sheludko, Z.M. Olevinskaya, N.Y. Spivak, N.V. Kuchuk // Цитология и генетика. — 2010. — Т. 44, № 5. — С. 60-64. — Бібліогр.: 32 назв. — англ. |
Institution
Digital Library of Periodicals of National Academy of Sciences of Ukraine| id |
nasplib_isofts_kiev_ua-123456789-66797 |
|---|---|
| record_format |
dspace |
| spelling |
nasplib_isofts_kiev_ua-123456789-667972025-02-23T17:52:51Z Production of human interferon alfa 2b in plants of Nicotiana excelsior by Agrobacterium-mediated transient expression Получение интерферона альфа-2b человека методом Agrobacterium-опосредованной транзиентной экспрессии в Nicotiana excelsior Отримання інтерферону альфа-2b людини методом Agrobacterіum-опосередкованої транзієнтної експресії в Nіcotіana excelsіor Sindarovska, Y.R. Gerasymenko, I.M. Sheludko, Y.V. Olevinskaya, Z.M. Spivak, N.Y. Kuchuk, N.V. Оригинальные работы Human interferon α2b gene was transiently expressed in Nicotiana excelsior plants. Fusion with N. plumbaginifolia calreticulin signal peptide for improved apoplast targeting and carrying out the expression under optimized conditions resulted in maximal interferon activity of 3.2 • 10³ IU/g fresh weight (FW) with an average of 2.1 ± 0.8 • 10³ IU/g FW. It proves that N. excelsior is a suitable host for Agrobacterium-mediated transient expression of genes encoding physiologically active human proteins. The transient expression conditions optimized for GFP marker protein were confirmed to be preferable for hIFN α2b. Ген интерферона α2b был транзиентно экспрессирован в растениях Nicotiana excelsior. Слияние целевого гена с последовательностью калретикулинового сигнального пептида из N. plumbaginifolia для улучшения транспорта продукта в апопласт и проведения транзиентной экспрессии в оптимальных условиях позволило добиться максимальной активности интерферона в листьях 3.2 • 10³ МЕ/г сырой массы при среднем значении 2.1 ± 0.8 • 10³ МЕ/г. Полученные результаты свидетельствуют о возможности использования N. excelsior для Agrobacterium-опосредованной транзиентной экспрессии фармацевтически активных белков человека. Показано, что условия транзиентной экспрессии, оптимизированные для получения репортерного белка GFP, подходят также для экспрессии гена интерферона α2b человека. Ген інтерферону α2b було транзієнтно експресовано у рослинах Nіcotіana excelsіor. Злиття цільового гена з послідовністю калретикулинового сигнального пептиду з N. plumbagіnіfolіa для поліпшення транспорту продукту в апопласт і проведення транзієнтної експресії в оптимальних умовах дозволило добитися максимальної активності інтерферону в листях 3.2 • 10³ МО/г сирої маси при середньому значенні 2.1 ± 0.8 • 10³ МО/г с.в. Отримані результати свідчать про можливість використання N. excelsіor для Agrobacterіum-опосередкованої транзієнтної експресії фармацевтично активних білків людини. Було показано, що умови транзієнтної експресії, оптимізовані для отримання репортерного білка GFP, підходять також для експресії гена інтерферону α2b людини. 2010 Article Production of human interferon alfa 2b in plants of Nicotiana excelsior by Agrobacterium-mediated transient expression / Y.R. Sindarovska, I.M. Gerasymenko, Y.V. Sheludko, Z.M. Olevinskaya, N.Y. Spivak, N.V. Kuchuk // Цитология и генетика. — 2010. — Т. 44, № 5. — С. 60-64. — Бібліогр.: 32 назв. — англ. 0564-3783 https://nasplib.isofts.kiev.ua/handle/123456789/66797 57.084.1+582.926.2+577.21 en Цитология и генетика application/pdf Інститут клітинної біології та генетичної інженерії НАН України |
| institution |
Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| collection |
DSpace DC |
| language |
English |
| topic |
Оригинальные работы Оригинальные работы |
| spellingShingle |
Оригинальные работы Оригинальные работы Sindarovska, Y.R. Gerasymenko, I.M. Sheludko, Y.V. Olevinskaya, Z.M. Spivak, N.Y. Kuchuk, N.V. Production of human interferon alfa 2b in plants of Nicotiana excelsior by Agrobacterium-mediated transient expression Цитология и генетика |
| description |
Human interferon α2b gene was transiently expressed in Nicotiana excelsior plants. Fusion with N. plumbaginifolia calreticulin signal peptide for improved apoplast targeting and carrying out the expression under optimized conditions resulted in maximal interferon activity of 3.2 • 10³ IU/g fresh weight (FW) with an average of 2.1 ± 0.8 • 10³ IU/g FW. It proves that N. excelsior is a suitable host for Agrobacterium-mediated transient expression of genes encoding physiologically active human proteins. The transient expression conditions optimized for GFP marker protein were confirmed to be preferable for hIFN α2b. |
| format |
Article |
| author |
Sindarovska, Y.R. Gerasymenko, I.M. Sheludko, Y.V. Olevinskaya, Z.M. Spivak, N.Y. Kuchuk, N.V. |
| author_facet |
Sindarovska, Y.R. Gerasymenko, I.M. Sheludko, Y.V. Olevinskaya, Z.M. Spivak, N.Y. Kuchuk, N.V. |
| author_sort |
Sindarovska, Y.R. |
| title |
Production of human interferon alfa 2b in plants of Nicotiana excelsior by Agrobacterium-mediated transient expression |
| title_short |
Production of human interferon alfa 2b in plants of Nicotiana excelsior by Agrobacterium-mediated transient expression |
| title_full |
Production of human interferon alfa 2b in plants of Nicotiana excelsior by Agrobacterium-mediated transient expression |
| title_fullStr |
Production of human interferon alfa 2b in plants of Nicotiana excelsior by Agrobacterium-mediated transient expression |
| title_full_unstemmed |
Production of human interferon alfa 2b in plants of Nicotiana excelsior by Agrobacterium-mediated transient expression |
| title_sort |
production of human interferon alfa 2b in plants of nicotiana excelsior by agrobacterium-mediated transient expression |
| publisher |
Інститут клітинної біології та генетичної інженерії НАН України |
| publishDate |
2010 |
| topic_facet |
Оригинальные работы |
| url |
https://nasplib.isofts.kiev.ua/handle/123456789/66797 |
| citation_txt |
Production of human interferon alfa 2b in plants of Nicotiana excelsior by Agrobacterium-mediated transient expression / Y.R. Sindarovska, I.M. Gerasymenko, Y.V. Sheludko, Z.M. Olevinskaya, N.Y. Spivak, N.V. Kuchuk // Цитология и генетика. — 2010. — Т. 44, № 5. — С. 60-64. — Бібліогр.: 32 назв. — англ. |
| series |
Цитология и генетика |
| work_keys_str_mv |
AT sindarovskayr productionofhumaninterferonalfa2binplantsofnicotianaexcelsiorbyagrobacteriummediatedtransientexpression AT gerasymenkoim productionofhumaninterferonalfa2binplantsofnicotianaexcelsiorbyagrobacteriummediatedtransientexpression AT sheludkoyv productionofhumaninterferonalfa2binplantsofnicotianaexcelsiorbyagrobacteriummediatedtransientexpression AT olevinskayazm productionofhumaninterferonalfa2binplantsofnicotianaexcelsiorbyagrobacteriummediatedtransientexpression AT spivakny productionofhumaninterferonalfa2binplantsofnicotianaexcelsiorbyagrobacteriummediatedtransientexpression AT kuchuknv productionofhumaninterferonalfa2binplantsofnicotianaexcelsiorbyagrobacteriummediatedtransientexpression AT sindarovskayr polučenieinterferonaalʹfa2bčelovekametodomagrobacteriumoposredovannojtranzientnojékspressiivnicotianaexcelsior AT gerasymenkoim polučenieinterferonaalʹfa2bčelovekametodomagrobacteriumoposredovannojtranzientnojékspressiivnicotianaexcelsior AT sheludkoyv polučenieinterferonaalʹfa2bčelovekametodomagrobacteriumoposredovannojtranzientnojékspressiivnicotianaexcelsior AT olevinskayazm polučenieinterferonaalʹfa2bčelovekametodomagrobacteriumoposredovannojtranzientnojékspressiivnicotianaexcelsior AT spivakny polučenieinterferonaalʹfa2bčelovekametodomagrobacteriumoposredovannojtranzientnojékspressiivnicotianaexcelsior AT kuchuknv polučenieinterferonaalʹfa2bčelovekametodomagrobacteriumoposredovannojtranzientnojékspressiivnicotianaexcelsior AT sindarovskayr otrimannâínterferonualʹfa2blûdinimetodomagrobacteríumoposeredkovanoítranzíêntnoíekspresíívnícotíanaexcelsíor AT gerasymenkoim otrimannâínterferonualʹfa2blûdinimetodomagrobacteríumoposeredkovanoítranzíêntnoíekspresíívnícotíanaexcelsíor AT sheludkoyv otrimannâínterferonualʹfa2blûdinimetodomagrobacteríumoposeredkovanoítranzíêntnoíekspresíívnícotíanaexcelsíor AT olevinskayazm otrimannâínterferonualʹfa2blûdinimetodomagrobacteríumoposeredkovanoítranzíêntnoíekspresíívnícotíanaexcelsíor AT spivakny otrimannâínterferonualʹfa2blûdinimetodomagrobacteríumoposeredkovanoítranzíêntnoíekspresíívnícotíanaexcelsíor AT kuchuknv otrimannâínterferonualʹfa2blûdinimetodomagrobacteríumoposeredkovanoítranzíêntnoíekspresíívnícotíanaexcelsíor |
| first_indexed |
2025-11-24T05:45:13Z |
| last_indexed |
2025-11-24T05:45:13Z |
| _version_ |
1849649385221128192 |
| fulltext |
УДК 57.084.1+582.926.2+577.21
Y.R. SINDAROVSKA 1, I.M. GERASYMENKO 1,
Y.V. SHELUDKO 1, Z.M. OLEVINSKAYA 2,
N.Y. SPIVAK 2, N.V. KUCHUK 1
1 Institute of Cell Biology and Genetic Engineering NAS of Ukraine, Kiev
03680, Ukraine,
E�mail: ysheludko@ukr.net
2 Institute of Microbiology and Virology NAS of Ukraine, Kiev
PRODUCTION OF HUMAN INTERFERON
ALFA 2b IN PLANTS OF NICOTIANA
EXCELSIOR BY AGROBACTERIUM�
MEDIATED TRANSIENT EXPRESSION
Human interferon α2b gene was transiently expressed in
Nicotiana excelsior plants. Fusion with N. plumbaginifolia
calreticulin signal peptide for improved apoplast targeting and
carrying out the expression under optimized conditions result�
ed in maximal interferon activity of 3.2 · 103 IU/g fresh weight
(FW) with an average of 2.1 ± 0.8 · 103 IU/g FW. It proves
that N. excelsior is a suitable host for Agrobacterium�mediat�
ed transient expression of genes encoding physiologically active
human proteins. The transient expression conditions optimized
for GFP marker protein were confirmed to be preferable for
hIFN α2b.
Introduction. Interferons are a large family of
multifunctional secreted proteins involved in ani�
mal antiviral defence, cell growth regulation and
immune system activation [1]. Interferons are used
to treat several diseases including some types of
cancer and hepatitis C [1]. Recombinant human
interferons have been produced in bacteria, yeast,
insect and mammalian cells, and several plant
species [2–11]. Plants as a source of foreign pro�
teins have a number of advantages over microbial
or animal cell systems. In contrast to bacteria cor�
rect posttranslational modifications of recombi�
nant proteins take place in plant cells as well as
folding and assembling of multimeric proteins, e.g.
antibodies [12–14]. Plants do not contain bacteri�
al toxins and human pathogens like viruses and
prions, that makes the recombinant proteins of
plant origin safer [15]. In some cases they can be
used without prior purification as edible vaccines
that lowers production costs considerably [15].
The main drawback of plants with stably trans�
formed nuclear genome is the moderate level of
target protein accumulation. The recombinant
protein production usually does not exceed of 1 %
of total soluble proteins (TSP) [16] due to the low
transgene expression level and/or the protein
product instability (for strategies to enhance stable
transgene expression and product accumulation in
plants, see [17]). Plastid transformation often
allows selecting of transplastomic plants with high
level of recombinant protein accumulation [18].
However, it is a time�consuming task, up to date
feasible for a restricted number of plant species.
Modern approaches to transient gene expression
in plants lead to accumulation of large amount of
recombinant proteins within a very short time [19].
This method was successfully applied for produc�
tion of a number of recombinant proteins, e.g.
tumor�specific antibodies [20] and vaccines [21]
and human growth hormone [22]. The efficiency
of the transient expression may be increased by
vector system modification [23, 24] and/or by opti�
mization of the expression conditions and choosing
of an appropriate plant host species [25].
Here we report the production of active human
interferon alfa 2b in plants of Nicotiana excelsior,
previously selected as a promising host species for
Agrobacterium�mediated transient expression using
GFP marker protein [25].
Materials and methods. Plant material. Seeds of
N. benthamiana and N. excelsior were obtained from
the National Germplasm Bank of World Flora of
ISSN 0564–3783. Цитология и генетика. 2010. № 560
© Y.R. SINDAROVSKA, I.M. GERASYMENKO, Y.V. SHELUDKO,
Z.M. OLEVINSKAJA, N.Y. SPIVAK, N.V. KUCHUK, 2010
the Institute of Cell Biology and Genetic Enginee�
ring (Kiev, Ukraine). In greenhouse plants were
grown at 20–25 °С and 14 h light period (3000–
4000 lux).
Bacterial strains and genetic constructs. Genetic
constructs pICH10881, pICH10570, pICH13301
(with the native hIFN�α2b gene), pICH17311 (with
the recombinant hIFN�α2b gene) and pICH7410
(with reporter GFP gene) represent a viral�based
module vector system described in details in [23].
The plasmid pICH6692 contained the gene of the
p19 protein of tomato bushy stunt virus, a suppres�
sor of post�transcriptional gene silencing [26] driven
by 35S CaMV promoter. All the mentioned plas�
mids were obtained for scientific purposes from
Icon Genetics GmbH (Halle/Saale, Germany).
Agrobacterium tumefaciens strain GV3101 trans�
formed with individual constructs was grown
overnight in LB medium supplemented with
50 mg/l of rifampicin and 50 mg/l of carbenicillin
or kanamycin, and 100 μM of acetosyringone.
Transient expression assay. Plant infiltration was
performed as described in [27] with several modi�
fications [23]: A. tumefaciens cells of overnight cul�
ture were centrifuged and resuspended with the
infiltration buffer (10 mM MES, pH 5.5; 10 mM
MgSO4; 100 μM acetosyringone). The Agrobacte�
rium suspensions harboring different plasmid vec�
tors were mixed in the equal volumes so that the
final optical density (OD 600) of each suspension
in the infiltration buffer amounted 0.5. The leaves
of greenhouse grown plants were infiltrated with
Agrobacterium mixture (50 mkl/leaf) by using a
syringe without a needle. The Agrobacterium mix�
ture was injected into mesophile tissue of the 2nd –
4th upper leaves. After infiltration, the plants were
further grown under greenhouse conditions and
harvested at 14–18 days post infiltration. All exper�
iments were carried out in 4–6 replications.
Interferon activity assay. Extracts from plant
leaves were prepared in equal volume of 100 mM
Tris/HCl buffer, pH 8.0, containing 5 mM
Na2EDTA, 100 mM NaCl, 10 mM β�mercap�
toethanol, and 2.5 % PVP. The total protein con�
tent was measured by the method of Bradford [28].
The assays were performed in sterile 96�well
microtiter plates. Each well was filled with 100 μl
of transmissible neonatal pig testis cell suspension
(105 cell/ml) in RPMI�1640 medium [31]. The
cells were cultivated for 18 hours at 37 °С. Samples
to be titrated for interferon activity were diluted
20�fold with RPMI�1640 medium and placed in the
first row of wells of an empty plate. Two�fold dilu�
tions of the samples with RPMI�1640 medium were
made serially to the end of the well columns. The
RPMI�1640 medium in the wells with the cells was
replaced with sample dilution rows and the cells
were further incubated for 18 hours at 37 °С. The�
reafter 100 μl of vesicular stomatitis virus (100
CPE50/0.1 ml) was added to each well (except of
cell control wells, where 100 μl of RPMI�1640
medium were applied). The cells were cultivated at
37 °С for 24 hours (until the cytopathic effect was
fully developed in the virus control wells). The
medium from the wells was evaporated and the
cells were stained with crystal violet. The wells
where 50 % of the cells were protected from the
virus cytopathic effect were detected by microscop�
ic examination. Standard interferon solution was
included in all assays to determine the absolute titer.
Results and discussion. Transient expression is
often carried out in N. benthamiana [e.g., 14, 22],
but this plant species has a rather small biomass
yield that hinders its application for large�scale
production of recombinant proteins. In our previ�
ous studies we have selected N. excelsior as a better
host for transient expression. This species dis�
played the best characteristics in regard to biomass
yield as well as GFP accumulation level [25].
The target hIFN�α2b gene was introduced into
N. excelsior plants as a part of a viral�based module
vector system. This system consists of three ele�
ments delivered in plant by simultaneous infiltra�
tion of Agrobacterium strains carrying correspon�
ding plasmids. Two modules containing viral genes
and regulatory elements and the target gene are
combined inside of the plant cell by the Streptomyces
phage PhiC31 site�specific recombinase encoded
in the third plasmid. The resulting DNA molecule
contains the viral genes of the RNA�dependent
RNA polymerase, movement protein and the tar�
get gene driven by the subgenomic promoter of a
viral coat protein. DNA is able to move from cell
to cell due to the movement protein [23].
In our experiments the greenhouse grown N.
excelsior plants were infiltrated with a mixture of
four Agrobacterium strains carrying the three mod�
ules of the viral�based expression system and a vec�
tor with the gene encoding p19 protein of tomato
bushy stunt virus, a suppressor of gene silencing
ІSSN 0564–3783. Цитология и генетика. 2010. № 5 61
Production of human interferon alfa 2b in plants of Nicotiana excelsior
[26]. For monitoring of the transient expression
process, one of the leaves on each plant was infiltrat�
ed with an analogous Agrobacterium mixture but
carrying the marker GFP gene instead of hIFN�
α2b. The activity of interferon in the leaf extracts
was measured by its ability to protect animal cells
in vitro against viral replication [29, 30]. For the
construct with native hIFN�α2b gene we deter�
mined interferon activity in the extracts and found
that it reached the maximum level of 8 · 102 IU/g
FW with an average of 1.3 ± 0.59 · 102 IU/g FW.
This value corresponds to 1.3–2 ng/g of leaf fresh
weight.
We checked several parameters which were
shown to influence on recombinant protein produc�
tion via transient expression using GFP as a reporter
[25]. It was found that the activity of interferon was
approximately 3–4 times higher in the upper leaves
of the plant than in lower ones (2.5 · 102 IU/g and
0.65 · 102 IU/g, respectively). Co�expression of
the p19 protein, a suppressor of gene silencing
[17], improved the interferon yield at least 15�fold
(8 · 102 IU/g compared with 0.5 · 102 IU/g in the
experiments without the p19 suppressor of silenc�
ing). These data corresponds well to the results
obtained for GFP reporter protein [25].
The accumulation level of interferon was consid�
erably lower than that of GFP obtained under the
same conditions [25]. These results can be explained
by lower stability of interferon in the plant cells as
compared with GFP. The action of cell proteases is
known to be a limiting factor for recombinant pro�
tein accumulation in plants [32]. One of possible
ways to overcome this problem is subcellular pro�
tein targeting to organelles (e.g. chloroplasts or
endoplasmic reticulum (ER)) or into apoplast [17,
22]. It is accomplished by fusion of the protein of
interest with specific aminoacid sequences that
direct it to the corresponding cell compartment.
To be excreted into apoplast, a protein should con�
tain on its N�terminus a signal sequence that is
usually cleaved during protein translocation through
the ER membrane. Native hIFN�α2b gene encodes
signal sequence that ensures its secretion from
leukocytes. This sequence supports analogous tar�
geting in plant cells, but possibly at lower rate.
To amend the process of interferon excretion
into apoplast, we have used a recombinant hIFN�
α2b gene attached to N. plumbaginifolia calretic�
ulin signal peptide. It was reported previously that
fusion with N. plumbaginifolia calreticulin signal
peptide led to high level of transient expression of
hIFN�α2b gene in N. benthamiana [22]. In our
experiments the transient expression of the recom�
binant hIFN�α2b gene resulted in approximately
15�fold higher interferon activity than in case of
using the native gene (2.06 ± 7.8 · 103 IU/g of leaf
extract corresponding to 20–30 ng/g FW) (Figure).
The maximum activity amounted to 3,2 · 103 IU/g
of leaf extract (30–50 ng/g FW).
Physiologically active human interferons have
been produced earlier in several plant species by
stable nuclear transformation [2–7], chloroplast
transformation [8] and transient expression [9–
11]. The highest level of hIFN�α2b (up to 20 %
TSP, or 3 mg/g FW) was reported for tobacco
transplastomic plants [8]. The interferon activity in
stable nuclear transformants reached approximate�
ly 5 · 102 IU/g FW [5, 11]. Transient expression
resulted in approximately 10�fold higher activities
(3.1 · 104 IU/ml for interferon β in lettuce [10] and
2.1 · 104 IU/g FW for interferon α2a in cucurbits
[9]), although in some cases the interferon con�
tent was lower (0.3 ng/g FW for chicken interferon
α in lettuce [11]). Our results with 2.1 ± 0.8 · 103 IU/g
FW of human interferon α2b in N. excelsior prove
the effectiveness of the transient expression
method.
We can conclude that N. excelsior is a suitable
host for transient expression of genes encoding
physiologically active human proteins, e.g. inter�
feron α2b. The transient expression conditions
optimized for GFP marker protein were con�
firmed to be preferable for hIFN α2b.
ISSN 0564–3783. Цитология и генетика. 2010. № 562
Y.R. Sindarovska, I.M. Gerasymenko, Y.V. Sheludko et al.
Activity of interferon in leaf extracts of N. excelsior transient�
ly expressing native or recombinant (fused to N. plumbagini�
folia calreticulin signal peptide) human interferon α2b gene
Я.Р. Синдаровская, И.М. Герасименко,
Ю.В. Шелудько, З.M. Олевинская,
Н.Я. Спивак, Н.В. Кучук
ПОЛУЧЕНИЕ ИНТЕРФЕРОНА АЛЬФА�2b
ЧЕЛОВЕКА МЕТОДОМ AGROBACTERIUM�
ОПОСРЕДОВАННОЙ ТРАНЗИЕНТНОЙ
ЭКСПРЕССИИ В NICOTIANA EXCELSIOR
Ген интерферона α2b был транзиентно экспресси�
рован в растениях Nicotiana excelsior. Слияние целевого
гена с последовательностью калретикулинового сиг�
нального пептида из N. plumbaginifolia для улучшения
транспорта продукта в апопласт и проведения транзи�
ентной экспрессии в оптимальных условиях позволило
добиться максимальной активности интерферона в
листьях 3.2 · 10
3
МЕ/г сырой массы при среднем зна�
чении 2.1 ± 0.8 · 10
3
МЕ/г. Полученные результаты
свидетельствуют о возможности использования N. excel�
sior для Agrobacterium�опосредованной транзиентной
экспрессии фармацевтически активных белков чело�
века. Показано, что условия транзиентной экспрессии,
оптимизированные для получения репортерного белка
GFP, подходят также для экспрессии гена интерферона
α2b человека.
Я.Р. Сіндаровська, І.М. Герасименко,
Ю.В. Шелудько, З. M. Олевинська,
М.Я. Співак, М.В. Кучук
ОТРИМАННЯ ІНТЕРФЕРОНУ АЛЬФА�2b
ЛЮДИНИ МЕТОДОМ AGROBACTERІUM�
ОПОСЕРЕДКОВАНОЇ ТРАНЗІЄНТНОЇ
ЕКСПРЕСІЇ В NІCOTІANA EXCELSІOR
Ген інтерферону α2b було транзієнтно експресовано
у рослинах Nіcotіana excelsіor. Злиття цільового гена з
послідовністю калретикулинового сигнального пепти�
ду з N. plumbagіnіfolіa для поліпшення транспорту про�
дукту в апопласт і проведення транзієнтної експресії в
оптимальних умовах дозволило добитися максимальної
активності інтерферону в листях 3.2 · 10
3
МО/г сирої
маси при середньому значенні 2.1 ± 0.8 · 10
3
МО/г с.в.
Отримані результати свідчать про можливість викори�
стання N. excelsіor для Agrobacterіum�опосередкованої
транзієнтної експресії фармацевтично активних білків
людини. Було показано, що умови транзієнтної екс�
пресії, оптимізовані для отримання репортерного білка
GFP, підходять також для експресії гена інтерферону
α2b людини.
REFERENCES
1. Chelbi�Alix M.K., Wietzerbin J. Interferon, a growing
cytokine family: 50 years of interferon research //
Biochimie. – 2007. – 89. – P. 713–718.
2. Edelbaum O., Stein D. et al. Expression of active human
interferon�beta in transgenic plants // J. Interferon
Res. – 1992. – 12. – P. 449–453.
3. Masumura T., Morita S. et al. Production of biological�
ly active human interferon�α in transgenic rice // Plant
Biotechnol. – 2006. – 23. – P. 91–97.
4. Chen T.L., Lin Y.L. et al. Expression of bioactive human
interferon�gamma in transgenic rice cell suspension
cultures // Transgenic Res. – 2004. – 13. – P. 499–510.
5. Ohya K., Matsumura T. et al. Expression of two subtypes
of human IFN�alpha in transgenic potato plants // J.
Interferon. Cytokine Res. – 2001. – 21. – P. 595–602.
6. Sawahel W.A. The production of transgenic potato plants
expressing human alpha�interferon using lipofectin�
mediated transformation // Cell Mol. Biol. Lett. –
2002. – 7. – P. 19–29.
7. Gerasymenko I.M., Lypova N.M., Sakhno L.A.,
Shcherbak N.L., Sindarovska Y.R., Bannikova M.A.,
Sheludko Y.V., Kuchuk N.V. Obtaining and analysis of
tоbассо, lettuce and rape plants transformed with
human interferon alfa 2b gene // Factors of
Experimental Evolution of Organisms / Ed. V.A.
Kunakh. – Kiev : Logos, 2009 – 7. – P. 274–279.
8. Arlen P.A., Falconer R. et al. Field production and func�
tional evaluation of chloroplast�derived interferon�alpha
2b // Plant Biotechnol. J. – 2007. – 5. – P. 511– 525.
9. Tzahi Arazi, Shalom Guy Slutsky, Yoel Moshe Shiboleth
et al. Engineering zucchini yellow mosaic potyvirus as a
non�pathogenic vector for expression of heterologous
proteins in cucurbits // J. Biotechnol. – 2001. – 87. –
P. 67–82
10. Li J., Chen M. et al. Transient expression of an active
human interferon�beta in lettuce // Sci. Hort. – 2007. –
112. – P. 258–265.
11. Song L., Zhao D.G. et al. Transient expression of chick�
en alpha interferon gene in lettuce // J. Zhejiang Univ.
Sci. B. – 2008. – 9. – P. 351–355.
12. Stoger E., Sack M., Fischer R., Christou P. Plantibodies :
Applications, advantages and bottlenecks // Curr. Opin
Biotechnol. – 2002. – 13. – P. 161–166.
13. Ma J.K., Drake P.M., Chargelegue D., Obregon P.,
Prada A. Antibody processing and engineering in
plants, and new strategies for vaccine production //
Vaccine. – 2005. – 23. – P. 1814–1818.
14. Huang Z., Phoolcharoen W., Lai H., Piensook K.,
Cardineau G., Zeitlin L., Whaley K.J., Arntzen C.J.,
Mason H.S., Chen Q. High�level rapid production of
full�size monoclonal antibodies in plants by a single�
vector DNA replicon system // Biotechnol Bioeng. –
2009. – 106. – P. 9–17.
15. Larrick J.W., Thomas D.W. Producing proteins in trans�
genic plants and animals // Curr. Opin. Biotechnol. –
2001. – 12. – P. 411–418.
16. Daniell H., Streatfield S.J., Wycoff K. Medical molecu�
lar farming: production of antibodies, biopharmaceuti�
cals and edible vaccines in plants // Trends in Plant
Sci. – 2001. – 6. – P. 219–226.
17. Streatfield S.J. Approaches to achieve high�level het�
ІSSN 0564–3783. Цитология и генетика. 2010. № 5 63
Production of human interferon alfa 2b in plants of Nicotiana excelsior
erologous protein production in plants // Plant
Biotechnol. J. – 2007. – 5. – P. 2–15.
18. Maliga P. Progress towards commercialization of plas�
tid transformation technology // Trends Biotechnol. –
2003. – 21. – P. 20–28.
19. Sheludko Y.V. Agrobacterium�mediated transient expres�
sion as an approach to production of recombinant pro�
teins in plants // Recent Patents on Biotechnology. –
2008. – 2. – P. 198–208.
20. Vaquero C., Sack M., Chandler J., Drossard J., Schuster F.,
Monecke M., Schillberg S., Fischer R. Transient expres�
sion of a tumor�specific single�chain fragment and a
chimeric antibody in tobacco leaves // Proc. Nat. Acad.
Sci. USA. – 1999. – 96, № 20. – P. 11128–11133.
21. McCormick A.A., Kumagai M.H., Hanley K. et al. Rapid
production of specific vaccines for lymphoma by
expression of the tumor�derived single�chain Fv epi�
topes in tobacco plants // Proc. Nat. Acad. Sci. USA. –
1999. – 96. – P. 703–708.
22. Gils M., Kandzia R., Marillonnet S., Klimyuk V., Gleba
Y. High�yield production of authentic human growth
hormone using a plant virus�based expression system //
Plant Biotechnol. J. – 2005. – 3. – P. 613–620.
23. Marillonnet S., Giritch A., Gils M., Kandzia R., Klimyuk V.,
Gleba Y. In planta engineering of viral RNA replicons:
efficient assembly by recombination of DNA modules
delivered by Agrobacterium // Proc. Nat. Acad. Sci. USA. –
2004. – 101. – P. 6852–6857.
24. Gleba Y., Klimyuk V., Marillonnet S. Magnifection–a
new platform for expressing recombinant vaccines in
plants // Vaccine. – 2005. – 23. – P. 2042–2048
25. Sheludko Y.V., Sindarovska Y.R., Gerasymenko I.M.,
Bannikova M.A., Kuchuk N.V. Comparison of several
Nicotiana species as hosts for high�scale Agrobacterium�
mediated transient expression // Biotechnol. Bioeng. –
2007. – 96. – P. 608–614.
26. Voinnet O., Rivas S., Mestre P., Baulcombe D. An enhanced
transient expression system in plants based on suppres�
sion of gene silencing by the p19 protein of tomato bushy
stunt virus // Plant J. – 2003. – 33. – P. 949–956.
27. Schob H., Kunz C, Meins F.Jr. Silencing of transgenes
introduced into leaves by agroinfiltration: a simple, rapid
method for investigating sequence requirements for gene
silencing // Mol. Gen. Genet. – 1997. – 256. – P. 581–
585.
28. Bradford M.M. A rapid and sensitive method for the
quantification of microgram quantities of protein uti�
lizing the principle of protein�dye binding // Anal.
Biochem. – 1976. – 72. – P. 248–254.
29. Rubinstein S., Familletti P.S., Pestka S. Convenient assay
for interferons // J. Virol. – 1981. – 37, № 5. – P. 755–
758.
30. Белоцкий С.М., Спивак Н.Я. Интерфероны: биоло�
гические и клинические эффекты. – К.: Фитосо�
циоцентр, 2006. – 288 с.
31. Moore G.E., Gerner R.E., Franklin H.A. Culture of nor�
mal human leukocytes // J. Amer. Med. Ass. – 1967. –
199. – P. 519–524.
32. Benchabane M., Goulet C., Rivard D., Faye L., Gomord V.,
Michaud D. Preventing unintended proteolysis in plant
protein biofactories // Plant Biotechnol. J. – 2008. –
6. – P. 633–648.
Received 16.03.10
ISSN 0564–3783. Цитология и генетика. 2010. № 564
Y.R. Sindarovska, I.M. Gerasymenko, Y.V. Sheludko et al.
|