Multiplex PCR assay for detection of human interferon alpha2b gene in transgenic plants
During the last decade interferons are regarded as potent candidates for generation of plant-based edible vaccines because of broad spectrum of antiviral activities and adjuvant properties. Establishment and certification of numerous interferon producing plant systems requests development of fast an...
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Інститут клітинної біології та генетичної інженерії НАН України
2012
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nasplib_isofts_kiev_ua-123456789-1264752025-02-23T17:35:09Z Multiplex PCR assay for detection of human interferon alpha2b gene in transgenic plants Мультиплексный ПЦР анализ присутствия гена интерферона альфа-2b в трансгенных растениях Мультиплексний ПЛР аналіз присутності гена интерферону альфа-2b в трансгенних рослинах Gerasymenko, I.M. Sakhno, L.O. Mazur, M.G. Sheludko, Y.V. Оригинальные работы During the last decade interferons are regarded as potent candidates for generation of plant-based edible vaccines because of broad spectrum of antiviral activities and adjuvant properties. Establishment and certification of numerous interferon producing plant systems requests development of fast and efficient multiplex PCR protocol for the transgene detection in GM plants. Here we represent a protocol for simultaneous amplification in one assay of fragments of hIFN alpha 2b gene and two control genes, namely virD1 of Agrobacterium tumefaciens and conservative region of plant actin gene. В последнее десятилетие интерфероны рассматриваются как перспективные кандидаты для получения из растений в виде съедобных вакцин, поскольку обладают широким спектром антивирусной активности и адъювантными свойствами. Создание и сертификация многочисленных растительных систем, продуцирующих рекомбинантный интерферон, делают актуальной разработку быстрого и эффективного протокола мультиплексной ПЦР для определения данного трансгена в генетически модифицированных растениях. В настоящей публикации мы приводим метод детекции гена человеческого интерферона альфа-2b в трансгенных растениях с помощью совместной амплификации в ходе одной реакции фрагментов гена hINTα2b и двух контрольных генов, virD1 Agrobacterium tumefaciens и консервативного участка гена актина растений. В останнє десятиліття інтерферони розглядаються як перспективні кандидати для отримання з рослин у вигляді їстівних вакцин оскільки, вони мають широкий спектр антивірусної активності й ад’ювантні властивості. Створення і сертифікація численних рослинних систем, які накопичують рекомбінантний інтерферон, роблять актуальною розробку швидкого й ефективного протоколу мультиплексної ПЛР для визначення даного трансгена в генетично модифікованих рослинах. В цій публікації ми наводимо метод детекції гена людського інтерферону альфа-2b у трансгенних рослинах за допомогою сумісної ампліфікації в ході однієї реакції фрагментів гена hINTα2b і двох контрольних генів, virD1 Agrobacterium tumefaciens і консервативної ділянки гена актину рослин. Supported by NASU grant, № UkrISTEI 0110U006061 and grant of GASIIU, № UkrISTEI 0111U007598. 2012 Article Multiplex PCR assay for detection of human interferon alpha2b gene in transgenic plants / I.M. Gerasymenko, L.O. Sakhno, M.G. Mazur, Y.V. Sheludko // Цитология и генетика. — 2012. — Т. 46, № 4. — С. 3-8. — Бібліогр.: 27 назв. — англ. 0564-3783 DOI: 10.3103/S009545271204007X https://nasplib.isofts.kiev.ua/handle/123456789/126475 57.085.1:57.088.3:577.218 en Цитология и генетика application/pdf Інститут клітинної біології та генетичної інженерії НАН України |
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Digital Library of Periodicals of National Academy of Sciences of Ukraine |
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English |
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Оригинальные работы Оригинальные работы |
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Оригинальные работы Оригинальные работы Gerasymenko, I.M. Sakhno, L.O. Mazur, M.G. Sheludko, Y.V. Multiplex PCR assay for detection of human interferon alpha2b gene in transgenic plants Цитология и генетика |
| description |
During the last decade interferons are regarded as potent candidates for generation of plant-based edible vaccines because of broad spectrum of antiviral activities and adjuvant properties. Establishment and certification of numerous interferon producing plant systems requests development of fast and efficient multiplex PCR protocol for the transgene detection in GM plants. Here we represent a protocol for simultaneous amplification in one assay of fragments of hIFN alpha 2b gene and two control genes, namely virD1 of Agrobacterium tumefaciens and conservative region of plant actin gene. |
| format |
Article |
| author |
Gerasymenko, I.M. Sakhno, L.O. Mazur, M.G. Sheludko, Y.V. |
| author_facet |
Gerasymenko, I.M. Sakhno, L.O. Mazur, M.G. Sheludko, Y.V. |
| author_sort |
Gerasymenko, I.M. |
| title |
Multiplex PCR assay for detection of human interferon alpha2b gene in transgenic plants |
| title_short |
Multiplex PCR assay for detection of human interferon alpha2b gene in transgenic plants |
| title_full |
Multiplex PCR assay for detection of human interferon alpha2b gene in transgenic plants |
| title_fullStr |
Multiplex PCR assay for detection of human interferon alpha2b gene in transgenic plants |
| title_full_unstemmed |
Multiplex PCR assay for detection of human interferon alpha2b gene in transgenic plants |
| title_sort |
multiplex pcr assay for detection of human interferon alpha2b gene in transgenic plants |
| publisher |
Інститут клітинної біології та генетичної інженерії НАН України |
| publishDate |
2012 |
| topic_facet |
Оригинальные работы |
| url |
https://nasplib.isofts.kiev.ua/handle/123456789/126475 |
| citation_txt |
Multiplex PCR assay for detection of human interferon alpha2b gene in transgenic plants / I.M. Gerasymenko, L.O. Sakhno, M.G. Mazur, Y.V. Sheludko // Цитология и генетика. — 2012. — Т. 46, № 4. — С. 3-8. — Бібліогр.: 27 назв. — англ. |
| series |
Цитология и генетика |
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3
Îðèãèíàëüíûå ðàáîòû
ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2012. ¹ 4
© I.M. GERASYMENKO, L.O. SAKHNO, M.G. MAZUR,
Y.V. SHELUDKO, 2012
УДК 57.085.1:57.088.3:577.218
I.M. GERASYMENKO, L.O. SAKHNO,
M.G. MAZUR, Y.V. SHELUDKO
Institute of Cell Biology and Genetic Engineering NAS of Ukraine, Kiev
E-mail: ysheludko@ukr.net
MULTIPLEX PCR ASSAY
FOR DETECTION OF HUMAN
INTERFERON ALPHA2b GENE
IN TRANSGENIC PLANTS
During the last decade interferons are regarded as
potent candidates for generation of plant-based edible
vaccines because of broad spectrum of antiviral activities
and adjuvant properties. Establishment and certification
of numerous interferon producing plant systems requests
development of fast and efficient multiplex PCR protocol
for the transgene detection in GM plants. Here we
represent a protocol for simultaneous amplification in one
assay of fragments of hIFN alpha 2b gene and two control
genes, namely virD1 of Agrobacterium tumefaciens and
conservative region of plant actin gene.
Introduction. Interferons (IFNs) represent an
important group of multifunctional proteins coor-
dinating a diverse array of cellular programs in
animal organisms. Being a part of the non-speci-
fic immune system, they participate in proces-
ses of antiviral defence, cell proliferation regula-
tion, signal transduction and immune response
modulation [1, 2]. Because of their immunomo-
dulatory effects IFNs are used in treatment of
a variety of diseases including several types of
cancer, hepatitis C infections, rheumatoid arthritis,
osteoporosis etc [1, 3, 4]. Special attention was
focused on IFNs as broad spectrum antivirals
for prevention or treatment of acute influenza
infections [5] and as a potent adjuvant that may
be used with both parenterally and mucosally
administered vaccines [6].
Plant based recombinant proteins demonstrate
correct posttranslational modifications, folding
and assembling to multimeric products, e.g.
antibodies [7–9]. Some differences in glyco-
sylation patterns of proteins produced in plant
and mammalian cells apparently caused no sig-
nificant effect on their antigen characteristic or
specificity [10] and may be overcome by expres-
sion in host of human glycosyltransferases [11].
Numerous examples of immune responses in
humans or animals caused by oral delivery of
plant-based vaccines were reviewed in recent
publications [12–14]. Up to now no clear evi-
dence were reported about possible pathogenic
action of plant viruses on humans, that makes the
recombinant proteins of plant origin safer.
All the mentioned above suggest perspectives
of IFN producing transgenic plants as edible vac-
cines (alone or together with other immunogenic
proteins). Apart from bacteria, yeast, insect and
mammalian expression systems, recombinant hu-
man interferons have been obtained in numerous
plant species [15–20], see also [21] for review. It
is evidently that further evolution of plant-based
vaccine technologies will extend the spectrum of
INF producing plants. It requests development of
fast and efficient Multiplex PCR (MPCR) pro-
tocol for the transgene detection in GM plants.
Here we represent a protocol for detection of hu-
man interferon alpha2b gene in transgenic plants
by simultaneous amplification in one assay of
fragments of hINT 2b and two control genes,
namely virD1 of Agrobacterium tumefaciens and
conservative region of plant actin gene. Because
PCR techniques as a method for GMO detec-
4
I.M. Gerasymenko, L.O. Sakhno, M.G. Mazur, Y.V. Sheludko
ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2012. ¹ 4
tion is commonly approved by the regulatory au-
thorities [22], the developed MPCR assay can
be recommended for certification of GM plants
harboring gene of human interferon alpha2b.
Materials and methods. Plant material. Trans-
genic plants of Nicotiana tabacum, N. benthamiana
and Brassica napus carring human interferon 2b
(hINF 2b) gene under control of 35S CaMV
promoter were obtained by Agrobacterium-me-
diated transformation using GV3101 strain with
pBIN19-derived binary vectors [23]. Transgenic
Nicotiana plants transformed with GFP gene and
rape plants carrying bovine cyp11A1 gene coding
for cytochrome P450scc were used as a source of
nontarget DNA.
DNA isolation. DNA was extracted and puri-
fied from the leaf material as described in [24].
The DNA concentration was measured by UV
absorption at 260 nm and the purity was evalu-
ated by the A260 nm/A280 nm ratio using the Bio-
Photometer («Eppendorf», Germany).
Primers and PCR conditions. The oligonucleo-
tide primers specific for the sequences given in
Table were purchased from Macrochim (Ukraine).
The PCR reactions were carried out in 2720
Thermal Cycler («Applied Biosystems», USA). A
standard PCR assay (in 10 l of 1 × PCR buffer
consisting of 67 mM Tris-HCl, pH 8.8, 16.6 mM
(NH4)2SO4, 0.01 % Tween 20, 2.5 mM MgCl2) con-
tained 1 g template plant DNA, 0.5 U Taq DNA
polymerase («Helicon», Russia), 0.5 mM dNTP,
and primers (Table) in concentration 0.25 M
each. The PCR was carried out under follow-
ing conditions: initial denaturation at 94 °C for
5 min, 30 cycles including denaturation at 94 °C
for 30 sec, annealing at 60 °C for 30 sec and
extension at 72 °C for 30 sec, followed by final
extension at 72 °C for 5 min.
Restriction endonuclease analysis. For restric-
tion endonuclease analysis PCR products were
precipitated with ethanol and digested with BglII
restriction endonuclease («Fermentas», Lithu-
ania) in reaction buffer recommended by the
manufacturer.
Agarose gel electrophoresis. The DNA fragments
were analysed by electrophoresis in 1 % (w/v)
agarose gel in 1×TAE buffer (40 mM Tris-ace-
tate, 1 mM EDTA) at 150 V for 45 min and vi-
sualized with ethidium bromide. The GeneRuler
100 bp DNA Ladder («Fermentas», Lithuania)
was used as DNA size marker.
Results and discussion. Specificity of primers for
hINF 2b detection. Two primer pairs were de-
signed for amplification of human interferon 2b
gene (Fig. 1). Native hINF 2b encodes the inter-
feron precursor with a signal sequence that de-
fines the secretion of the mature interferon from
leukocytes and is cleaved during protein translo-
cation across the endoplasmic reticulum mem-
brane. This sequence supports the same transport
in plant cells, but it was reported that substitution
of native signal peptide with analogous sequence
of plant origin (e.g. derived from calreticulin gene
of N. plumbaginifolia) leads to significant increase
of recombinant interferon levels in plants [19,
27]. Both designed primer pairs are expected to
anneal to the part of hINF 2b coding for mature
interferon that makes them appropriate for de-
tection of native as well as recombinant hINF 2b
genes fused with different signal sequences. Spec-
List of primer pairs employed for PCR assays
Target sequence
NCBI
acc. no. Primer sequences (5'-3')
Amplicon
size (bp) References
hINF 2b
hINF 2b
Actin gene of Nicotiana tabacum
virD1
NT_008413
NT_008413
X63603
M17989
IntFor ctcctgcttgaaggacag
IntRev ggagtcctccttcatcag
IntFor4 ttgatgctcctggcacag
IntRev2 ttctgctctgacaacctc
For tttgctggagatgatgc
Rev cttgaatggcgacatac
For atgtcgcaaggcagtaagccca
Rev ggagtctttcagcatggagcaa
265
396
351
432
This study
This study
[25]
[26]
5
Multiplex PCR assay for detection of human interferon alpha2b genein transgenic plants
ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2012. ¹ 4
ificity of the primer pairs was checked against the
GenBank, EMBL and DDBJ databases using the
Primer-BLAST algorithm. The designed primer
pairs demonstrate homology to interferon genes
of animal origin and no significant similarity with
any plant genes that proves their suitability for
analysis of transgenic plants.
The simplex PCR assay with the target DNA
isolated from transgenic N. tabacum, N. ben-
thamiana and B. napus showed specific hINF 2b
amplicons of the expected size, i.e. 265 bp wit h
IntFor–IntRev primer pair (results not shown)
and 396 bp with IntFor4–IntRev2 oligonucle-
otides (Fig. 2). The leaves of nontransformed
plants of corresponding species served as negative
controls. To confirm the amplification specific-
ity, the primer set was used in PCR assay with
nontarget tobacco and N. benthamiana DNA
(containing GFP gene) and nontarget rape DNA
(with bovine cyp11A1 gene), where no amplifica-
tion was observed. However, further experiments
revealed that the primer pair IntFor4–IntRev2
was less efficient in multiplex PCR (results not
shown). The multiplex PCR assay was developed
using the IntFor–IntRev primer pair.
Additional primer pairs for multiplex PCR analy-
sis. It is advisable to apply for analysis of transgenic
plants a multiplex PCR system that includes two
control primer pairs. A pair for amplification of an
intrinsic plant gene serves as an internal positive
control. It allows to reveal false negative results
that are due to insufficient DNA quality. Another
primer pair should be complement to one of the
Agrobacterium virulence (vir) genes. These genes
are necessary for T-DNA transfer into a plant cell
but are not transferred themselves. Plant genetic
transformation is often carried out using Agrobac-
terium, so the PCR analysis of the primary trans-
formants may show false positive signals derived
from the residual agrobacterial contamination of
the plant tissues. Amplification of the vir gene
fragments indicates such false positive results.
Fig. 1. Scheme of human interferon 2b gene. Arrows indicate the primer positions
Fig. 2. Detection of hINT 2b gene in GM Nicotiana
tabacum, N. benthamiana and Brassica napus using the
primer pair IntFor4-IntRev2. Lane M – 100 bp DNA
ladder. The numbers at the top indicate the template
DNA used in each lane: 1 – no template; 2 – non-
GM tobacco; 3 – GM tobacco carring GFP gene; 4 –
GM tobacco carring hINT 2b gene; 5 – non-GM
N. benthamiana; 6 – GM N. benthamiana carring
GFP gene; 7 – GM N. benthamiana carring hINT 2b
gene; 8 – non-GM rape; 9 – GM rape carring cy-
p11A1 gene; 10 – GM rape carring hINT 2b gene
Fig. 3. Triplex PCR detection of hINT 2b gene in GM
N. tabacum, Brassica napus (a) and N. benthamiana
(b). Lane M, 100 bp DNA ladder. The numbers at the
top indicate the template DNA used in each lane: 1 –
non-GM tobacco; 2 – GM tobacco carring GFP gene;
3 – GM tobacco carring hINT 2b gene; 4 – no tem-
plate; 5 – A. tumefaciens GV3101; 6 – non-GM rape;
7 – GM rape carring cyp11A1 gene; 8 – GM rape
carring hINT 2b gene; 9 – non-GM N. benthamiana;
10 – GM N. benthamiana carring GFP gene; 11 – GM
N. benthamiana carring hINT 2b gene
6
I.M. Gerasymenko, L.O. Sakhno, M.G. Mazur, Y.V. Sheludko
ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2012. ¹ 4
We have included in the presented PCR assay
the primer pairs for amplification of 351 bp frag-
ment of actin gene and 432 bp fragment of virD1
gene of GV3101 strain of A. tumefaciens. These
primers were earlier successfully used in MPCR
system for detection of recombinant desaturase-
lichenase genes in transgenic tobacco, rape and
potato plants [25]. Although the primers for am-
plification of actin gene fragment were designed
using the tobacco sequence, they are comple-
ment to the conserved part of the gene and can
be used for analysis of different plant species. We
have examined N. tabacum, N. benthamiana and
B. napus plants with the developed triplex PCR
system. The hINF 2b amplicon of 265 bp was
observed only with target DNA from plants car-
rying hINF 2b gene, no amplification was de-
tected in non-target transgenic or non-transgenic
samples. The 351 bp fragment of actin gene was
amplified from all the samples of plant DNA,
whereas the 432 bp fragment of virD1 gene was
detected only if the DNA of A. tumefaciens was
used as template (Fig. 3).
Confirmation of the amplicon identity. The
amplified hINF 2b fragment was subjected to
restriction endonuclease hydrolysis in order to
prove its identity. The BglII enzyme was expected
to produce fragments of 105 and 159 bp from the
265 bp amplicon (Fig. 1). The experimental data
confirmed the identity of the hINF 2b fragments
obtained from the transgenic tobacco and rape
DNA (Fig. 4).
Limit of detection. It is recommended to use
0.5–1 g of total plant DNA in an assay for
transgene presence [24]. The developed MPCR
system is able to detect hINF 2b when 10 ng of
transgenic plant DNA is spiked with 1 g of non-
transgenic DNA that means 1 % of transgenic
DNA (Fig. 5). The virD1 gene is amplified if 5 ng
of total A. tumefaciens DNA is mixed with 1 g of
transgenic plant DNA (Fig. 6).
Fig. 4. Restriction fragments of 264 bp hINT 2b ampli-
con. Lane M, 100 bp DNA ladder; 1 – BglII digested
hINT 2b PCR product of tobacco DNA; 2 – undigest-
ed hINT 2b PCR product of tobacco DNA; 3 –
undigested hINT 2b PCR product of rape DNA; 4 –
BglII digested hINT 2b PCR product of rape DNA
(actin amplicon of 351 bp remains undigested)
Fig. 5. Detection of hINT 2b gene in transgenic N.
benthamiana DNA spiked with non transgenic DNA.
Lane M, 100 bp DNA ladder. The numbers at the top
indicate the template DNA used in each lane: 1 –
1 g of transgenic DNA; 2 – 100 ng of transgenic DNA
mixed with 900 ng of non transgenic DNA; 3 – 10 ng
of transgenic DNA mixed with 1 g of non transgenic
DNA; 4 – 1 ng of transgenic DNA mixed with 1 g of
non transgenic DNA; 5 – 0.1 ng of transgenic DNA
mixed with 1 g of non transgenic DNA; 6 – no tem-
plate
Fig. 6. Detection of Agrobacterium contamination in
transgenic plant DNA. Lane M, 100 bp DNA ladder.
The numbers at the top indicate the template DNA
used in each lane: 1 – 1 g of transgenic tobacco
DNA; 2 – 1 g of transgenic tobacco DNA mixed
with 5 ng of A. tumefaciens GV3101 DNA; 3 – 1 g
of transgenic tobacco DNA mixed with 1 ng of A. tu-
mefaciens GV3101 DNA; 4 – 5 ng of A. tumefaciens
GV3101 DNA
7
Multiplex PCR assay for detection of human interferon alpha2b genein transgenic plants
ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2012. ¹ 4
Conclusions. The described MPCR assay for
detection of hINF 2b gene can be applied in
course of selection of transformed plants, espe-
cially for screening of primary transformants be-
cause it allows for detection of Agrobacterium
contamination. Development of the MPCR met-
hod for hINF 2b detection is also a necessary
prerequisite for legalization of GM plants with
antiviral activity carrying this transgene [22].
Supported by NASU grant, ¹ UkrISTEI
0110U006061 and grant of GASIIU, ¹ UkrISTEI
0111U007598.
È.Ì. Ãåðàñèìåíêî, Ë.À. Ñàõíî,
Ì.Ã. Ìàçóð, Þ.Â. Øåëóäüêî
ÌÓËÜÒÈÏËÅÊÑÍÛÉ ÏÖÐ ÀÍÀËÈÇ
ÏÐÈÑÓÒÑÒÂÈß ÃÅÍÀ ÈÍÒÅÐÔÅÐÎÍÀ
ÀËÜÔÀ-2b  ÒÐÀÍÑÃÅÍÍÛÕ ÐÀÑÒÅÍÈßÕ
 ïîñëåäíåå äåñÿòèëåòèå èíòåðôåðîíû ðàññìà-
òðèâàþòñÿ êàê ïåðñïåêòèâíûå êàíäèäàòû äëÿ
ïîëó÷åíèÿ èç ðàñòåíèé â âèäå ñúåäîáíûõ âàêöèí,
ïîñêîëüêó îáëàäàþò øèðîêèì ñïåêòðîì àíòèâè-
ðóñíîé àêòèâíîñòè è àäúþâàíòíûìè ñâîéñòâàìè.
Ñîçäàíèå è ñåðòèôèêàöèÿ ìíîãî÷èñëåííûõ ðàñòè-
òåëüíûõ ñèñòåì, ïðîäóöèðóþùèõ ðåêîìáèíàíòíûé
èíòåðôåðîí, äåëàþò àêòóàëüíîé ðàçðàáîòêó áûñ-
òðîãî è ýôôåêòèâíîãî ïðîòîêîëà ìóëüòèïëåêñíîé
ÏÖÐ äëÿ îïðåäåëåíèÿ äàííîãî òðàíñãåíà â ãå-
íåòè÷åñêè ìîäèôèöèðîâàííûõ ðàñòåíèÿõ.  íàñ-
òîÿùåé ïóáëèêàöèè ìû ïðèâîäèì ìåòîä äåòåêöèè
ãåíà ÷åëîâå÷åñêîãî èíòåðôåðîíà àëüôà-2b â òðàíñ-
ãåííûõ ðàñòåíèÿõ ñ ïîìîùüþ ñîâìåñòíîé àìïëè-
ôèêàöèè â õîäå îäíîé ðåàêöèè ôðàãìåíòîâ ãåíà
hINT 2b è äâóõ êîíòðîëüíûõ ãåíîâ, virD1 Agro-
bacterium tumefaciens è êîíñåðâàòèâíîãî ó÷àñòêà
ãåíà àêòèíà ðàñòåíèé.
².Ì. Ãåðàñèìåíêî, Ë.Î. Ñàõíî,
Ì.Ã. Ìàçóð, Þ.Â. Øåëóäüêî
ÌÓËÜÒÈÏËÅÊÑÍÈÉ ÏËÐ ÀÍÀ˲Ç
ÏÐÈÑÓÒÍÎÑÒ² ÃÅÍÀ ÈÍÒÅÐÔÅÐÎÍÓ
ÀËÜÔÀ-2b  ÒÐÀÍÑÃÅÍÍÈÕ ÐÎÑËÈÍÀÕ
 îñòàííº äåñÿòèë³òòÿ ³íòåðôåðîíè ðîçãëÿ-
äàþòüñÿ ÿê ïåðñïåêòèâí³ êàíäèäàòè äëÿ îòðèìàííÿ
ç ðîñëèí ó âèãëÿä³ ¿ñò³âíèõ âàêöèí, îñê³ëüêè âîíè
ìàþòü øèðîêèé ñïåêòð àíòèâ³ðóñíî¿ àêòèâíîñò³ é
àä’þâàíòí³ âëàñòèâîñò³. Ñòâîðåííÿ ³ ñåðòèô³êàö³ÿ
÷èñëåííèõ ðîñëèííèõ ñèñòåì, ÿê³ íàêîïè÷óþòü ðå-
êîìá³íàíòíèé ³íòåðôåðîí, ðîáëÿòü àêòóàëüíîþ ðîç-
ðîáêó øâèäêîãî é åôåêòèâíîãî ïðîòîêîëó ìóëüòè-
ïëåêñíî¿ ÏËÐ äëÿ âèçíà÷åííÿ äàíîãî òðàíñãåíà
â ãåíåòè÷íî ìîäèô³êîâàíèõ ðîñëèíàõ.  ö³é ïó-
áë³êàö³¿ ìè íàâîäèìî ìåòîä äåòåêö³¿ ãåíà ëþä-
ñüêîãî ³íòåðôåðîíó àëüôà-2b ó òðàíñãåííèõ ðîñ-
ëèíàõ çà äîïîìîãîþ ñóì³ñíî¿ àìïë³ô³êàö³¿ â õîä³
îäí³º¿ ðåàêö³¿ ôðàãìåíò³â ãåíà hINT 2b ³ äâîõ
êîíòðîëüíèõ ãåí³â, virD1 Agrobacterium tumefaciens ³
êîíñåðâàòèâíî¿ ä³ëÿíêè ãåíà àêòèíó ðîñëèí.
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Received 17.10.11
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/ENU (Use these settings to create Adobe PDF documents best suited for high-quality prepress printing. Created PDF documents can be opened with Acrobat and Adobe Reader 5.0 and later.)
>>
/Namespace [
(Adobe)
(Common)
(1.0)
]
/OtherNamespaces [
<<
/AsReaderSpreads false
/CropImagesToFrames true
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(InDesign)
(4.0)
]
/OmitPlacedBitmaps false
/OmitPlacedEPS false
/OmitPlacedPDF false
/SimulateOverprint /Legacy
>>
<<
/AddBleedMarks false
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/ConvertColors /ConvertToCMYK
/DestinationProfileName ()
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/FlattenerPreset <<
/PresetSelector /MediumResolution
>>
/FormElements false
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/PreserveEditing true
/UntaggedCMYKHandling /LeaveUntagged
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/UseDocumentBleed false
>>
]
>> setdistillerparams
<<
/HWResolution [2400 2400]
/PageSize [612.000 792.000]
>> setpagedevice
|