Page 114 - Zbornik_Konf_2019

https://doi.org/10.15414/2019.9788055220703

4 International Scientific Conference Abstracts Book
th
'GREEN' SYNTHESIS OF HUMAN INTERFERON‐α2B IN 'HAIRY ROOT' CULTURE
OF ARTEMISIA TILESII PLANTS
Nadiia Matvieieva , Oleksandra Likhova ,Anatolij Shakhovsky , Yurij Kudriavets 2
1
1
2
1 Institute of Cell Biology and Genetic Engineering NAS of Ukraine, Kyiv, Ukraine;
E-mail.: joyna@ukr.net
2 R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology NAS of Ukraine,
Kyiv, Ukraine
Agrobacterium rhizogenes – mediated transformation of plants is the method for foreign
genes transferring to plant genome. This process results in “hairy” roots formation and is a
way to change plant genome since bacterial cells can carry not only its own Ri–plasmid but
also plasmids with a wide range of target genes including mammalian origin genes. Study of
the possibility of human interferon-α2b ifn‐α2b gene transfer and synthesis of the protein in
Artemisia tilesii Ledeb. “hairy” root cultures was the aim of the work.
Artemisia tilesii “hairy” root cultures were obtained using the transformation by A.
rhizogenes A4 carried pCB124 and pCB161 plasmids with human ifn‐α2b gene (35S and Mll
promotors respectively). Roots were subcultured on the agar-solidified ½ MS medium (20 g/L
sucrose, pH 5.7). Polymerase chain reaction combined with reverse transcription (RT-PCR)
was performed to study the transcription of transferred ifn‐α2b gene in transformed root
lines. Antiviral activity of the extracts was determined using a micro method to reduce the
cytopathic effect of vesicular stomatitis virus Indiana strain (VSV) in the MDBK kidney bovine
cells highly sensitive to the antiviral effect of human IFN-alpha. The international standard
interferon-alpha (2nd WHO International Standard 1999 Human Interferon alpha 2b, rDNA E.
coli derived 95/566, 70000 IU per ampoule) was used as a standard in the investigation.
Extracts from the control roots (the plants were cultivated in in vitro conditions) did not
inhibit VSV. RT-PCR analysis demonstrated the presence of mRNA in all samples of “hairy”
roots studied. At the same time, the differences in the level of antiviral activity of the extracts
from some “hairy” root lines were founded. For example, the antiviral activity of extracts from
transgenic roots obtained using vector pCB124 with human ifn‐α2b gene under the control of
35S cauliflower mosaic virus promoter significantly exceeded the activity of the extracts from
transgenic roots obtained by the transformation using pSB161 vector with human ifn‐α2b
gene under the control of Mll promotor. It must be noted that in the extract of one line, despite
the presence of mRNA according to RT-PCR analysis, there was no activity against VSV. It may
be due to problems in the translation process, post-translational changes or because of a very
small amount of the protein synthesized in the root cells. Analysis of antiviral activity revealed
that extracts from the “hairy” root lines (vector pCB124) possessed antiviral activity against
VSV within 652 ... 98437 IU / g wet weight or 0.2 ... 28.0 IU / mg of total soluble protein.
So, the high antiviral activity founded in the extracts of A. tilesii transgenic roots
indicates a great biotechnological potential of these plants.
Keywords: Artemisia tilesii Ledeb., “hairy” root culture, human interferon-α2b, antiviral activity.

|114 4 International Scientific Conference Agrobiodiversity Nutrition, Health and Quality of Human and Bees Life
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September 11–13, 2019