APPLICATION OF BIOTECHNOLOGY IN PLANT-BASED VACCINE PRODUCTION AGAINST HERPES SIMPLEX VIRUS
AYA HUSSEIN MUSTAFA AL-TURKEY;
Abstract
Herpes simplex virus (HSV) is a DNA-viral genome and causing infection for both human and animal. HSV-2 is a sexually-transmitted virus. It is prevalence in European countries and also in Egyptian-closed societies i.e. in Upper Egypt.
Egyptian isolate of HSV-2 was isolated and its 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 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 sub-cloned 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 were transformed into Agrobacterium for Agro-inoculation. Explants of Castell Rock and MPI tomato varieties were inoculated with the HSV-2gD-PB21 binary vector-treated 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.
Key words:
Herpes simplex virus 2 –Polymerase chain reaction (PCR) – cloning – agro-inoculation–Agrobacterium Transformation - edible vaccine – tomato tissue culture – oral vaccines – gene expression – sexual transmitted disease.
Egyptian isolate of HSV-2 was isolated and its 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 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 sub-cloned 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 were transformed into Agrobacterium for Agro-inoculation. Explants of Castell Rock and MPI tomato varieties were inoculated with the HSV-2gD-PB21 binary vector-treated 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.
Key words:
Herpes simplex virus 2 –Polymerase chain reaction (PCR) – cloning – agro-inoculation–Agrobacterium Transformation - edible vaccine – tomato tissue culture – oral vaccines – gene expression – sexual transmitted disease.
Other data
| Title | APPLICATION OF BIOTECHNOLOGY IN PLANT-BASED VACCINE PRODUCTION AGAINST HERPES SIMPLEX VIRUS | Other Titles | تطبيق التكنولوجيا الحيويه فى إنتاج لقاح عن طريقالنبات ضد فيروس الهربس البسيط | Authors | AYA HUSSEIN MUSTAFA AL-TURKEY | Issue Date | 2015 |
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