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العنوان
APPLICATION OF BIOTECHNOLOGY IN PLANTBASED
VACCINE PRODUCTION AGAINST HERPES
SIMPLEX VIRUS /
المؤلف
AL-TURKEY,AYA HUSSEIN MUSTAFA.
هيئة الاعداد
باحث / AYA HUSSEIN MUSTAFA AL-TURKEY
تاريخ النشر
2015
عدد الصفحات
149p.;
اللغة
الإنجليزية
الدرجة
ماجستير
التخصص
الزراعية والعلوم البيولوجية (المتنوعة)
تاريخ الإجازة
1/1/2015
مكان الإجازة
جامعة عين شمس - كلية الزراعة - فيروسات زراعيه
الفهرس
Only 14 pages are availabe for public view

from 149

from 149

Abstract

Herpes simplex virus (HSV) is a DNA-viral genome and cause infection for
both human and animal. HSV-1 and HSV-2 which are two members of the
herpes virus family, Herpesviridae, are causing infection to human and was
considered as a sexually-transmitted virus. HSV-2 is prevalence in
European countries and also in Egyptian-closed societies i.e. in Upper
Egypt.Children in Egypt with acute lymphoblastic leukemia are often
infected with HSV from a young age—HSV-2 antibodies are present in an
estimated 54% of children under the age of 5, and 77% in children over 10
years of age Algerian children are also likely to acquire HSV-1 infection at
a young age (under 6) and 81.25% of the population has antibodies to HSV-
1 by the age of 15. Genital HSV-2 infection is one of important sexually
transmitted diseases all over the world. More than half billion of people are
infected with HSV-2, and nearly 24 millions of new cases are added per
year.Herpes simplexvirus (HSV) infections of the skin and Coetaneous
Herpes simplex is characterized by painful, burning, or clusters of vesicles
on the lips, oral mucous membranes, genital region, genital ulcers or other
areas of the body. HSV infection of the eye results in keratoconjunctivitis, a
serious condition that sometimes leads to corneal blindness. HSV may also
cause encephalitis or other systemic infections, particularly in
immunocompromised patients.
Tomato (Lycopersicon esculentum Mill.) is an important solanaceous
vegetable crop grown throughout the world for its versatile uses. It is a
major vegetable crop that has achieved tremendous popularity over the last
century. It is one of the most important protective foods as it possesses
appreciable quantities of vaitamins and minerals and sometime rightly
referred to as poor man’s orange. Tomato is normally grown from hybrid
seeds. Hybrid seeds are expensive to produce due to their reliance on
manual labour. Alternative methods are therefore needed to minimise the
cost of production of seedlings. Tissue culture techniques have the potential
to meet these requirements.In vitro techniques are important tools for modern plant improvement programs to produce virus free plants, to
introduce new traits into selected plants, to multiply elite selections, and to
develop suitable cultivars in the minimum time. Tomatoes breeding
programs can highly benefit of biotechnological tools, such as gene transfer
technology, which allows the introduction of foreign genes into a
germplasm, without modifying the genetic background of elite varieties.
However, a breeding program associated to biotechnological tools depends
upon the development of an efficient in vitro plant regeneration system.
Efficient plantlet regeneration in tomato was reported from meristems, leaf,
stems, anthers and hypocotyls. In vitro plant regeneration of tomatoes using
protocols for adventitious shoot regeneration from cotyledon segments has
been reported. The system is based on three culture steps: a bud induction
phase, culturing the explants in medium supplemented with cytokinin an
elongation phase, transferring the shoot buds to medium with a lower
concentration of cytokinin and a rooting phase, using a culture medium
supplemented with auxin.
Plant engineering technology is now at an exciting stage of
development and the use of plants to produce vaccine antigens for human
diseases is a novel and promising system with several practical advantages
compared to fermentation or cell-culture facilities. Explants of Castle Rock
and MPI tomato varieties were transferred into regeneration medium and
Agro-inoculation was done using heat shock technique for HSV-2gD-PB21
binary vector-treated Agrobacterium competent cells. Agro-inoculated
tomato plants were tested for HSV-2gD insert occurrence by RT_PCR and
the results was successful in getting insert-containing tomatoes against HSV-
2 and the aim of the study is done.
The research was described the development of an experimental plantderived
subunit vaccine against HSV-2 produced in transgenic plants using
the PBI 121as a binary vector. Egyptian isolate of HSV-2 was isolated and Glycoprotein D (gD) subunit
was amplified, cloned and sequenced for characterization and detection.
HSV-2gD subunit was amplified, using specific forward and reverse primers,
with 1021 bp fragment size. The PCR product was molecularly cloned in
One Shot Top 10 chemically competent E.coli cells using PCR 2.1/TOPO/
TA cloning vector. DNA insert was liberated from 1 μg recombinant plasmid
by using the restriction endonuclease EcoR1. HSV-2 insert was successfully
subcloned by ligation intopurified binary vector PBI 121, using restriction
endonucleases XbaI and T4 DNA ligase enzyme and transformed in DH5α
competent E.coli cells.
Agrobacterium tumifaciens LBA 4404 strain competent cells were prepared.
HSV-2gD subunit-containing binary vector PBI 121 wAS transformed into
Agrobacterium for Agro-inoculation. Explants of Castell Rock and MPI
tomato varieties were inoculated with the HSV-2gD-PB21 binary vectortreated
Agrobacterium competent cells. Agro-inoculated tomato plants were
tested for HSV-2gD insert occurrence by PCR. The expression of gD was
confirmed through the RT_PCR for the messenger RNA as well as ELISA
for the expressed protein. The results were successful in getting transgenic
tomatoes expressing the HSV-2gD as a step forward to develop an edible
plant vaccine which is the aim of the study.