الفهرس | Only 14 pages are availabe for public view |
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. |