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Study on Fusion Protein and Its gene in Baculovirus Specificity BY Waleed Abdel-Gaber Helmy Nemr B.sc. (Biotechnology) Fac. Agriculture, Cairo Univ., 2002 Molecular Biology Diploma GEBRI, 2006 THESIS Submitted in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE In Genetic Engineering and Biotechnology (Animal tissue and cell culture) Department of Animal Biotechnology Genetic Engineering and Biotechnology Research Institute (GEBRI) Minufiya University EGYPT 2012 APPROVAL SHEET Study on Fusion Protein and Its gene in Baculovirus Specificity Msc Thesis In Genetic engineering and biotechnology “Animal tissue and cell culture” BY Waleed Abdel-Gaber Helmy Nemr B.sc. (Biotechnology) Fac. Agriculture, Cairo Univ., 2002 Molecular Biology Diploma GEBRI, 2006 APPROVAL COMMITTEE Dr. Khaled Dougdoug ………………………………. Professor of virology, Fac. Agric. Ain Shams University. Dr. Naglaa Al- Sinary ………………………………. Professor of natural products, National Center for Research and Radiation Technology, Atomic Energy Authority. Dr. Omaima A. Khamiss ……………………………… Associate Prof. of Animal Cell Culture and Virology, Animal Biotechnology Dep., GEBRI, Minufiya University. Study on Fusion Protein and Its gene in Baculovirus Specificity BY Waleed Abdel-Gaber Helmy Nemr B.sc. (Biotechnology) Fac. Agriculture, Cairo Univ., 2002 Molecular Biology Diploma GEBRI, 2006 For the master degree of Genetic engineering and biotechnology “Animal tissue and cell culture” Animal Biotechnology Department Genetic Engineering and Biotechnology Research Institute (GEBRI) Minufiya University Supervision Committee Dr. Omaima A. Khamiss …………………… Associate Prof. of Animal Cell Culture and Virology, Animal Biotechnology Dep., GEBRI, Minufiya University. Prof. Dr. Abdel-Moneam S. Bashandy …………………... Prof. of Radiation Microbiology, National Center for Research and Radiation Technology, Atomic Energy Authority. Dr. Adly Abd-Allah ………………….. Associate Prof., Department of Virology, National Research Center (NRC) ABSTRACT Baculoviruses are subdivided into two groups depending on the type of budded virus envelop fusion protein; group I utilized gp64 which include the most of nucleopolyhedroviruses (NPVs), group II utilized F protein which include the remnants of NPVs and all Granuloviruses (GVs). Recent studies reported the viral F protein coding gene as a host cellular sourced gene and may evolutionary acquired from the host genome referring to phylogeny analysis of fusion proteins. Thus, it was deduced that F protein coding gene is species- specific nucleotide sequence related to the type of the specific host and if virus could infect an unexpected host, the resulted virus may encode a vary F gene. In this regard, the present study utilized the mentioned properties of F gene in an attempt to produce a model of specific and more economic wider range granulovirus bio- pesticide able to infect both Spodoptera littoralis and Phthorimaea operculella larvae. Multiple sequence alignment and phylogeny analysis were performed on six members of group II baculovirus, novel universal PCR primers were manually designed from the conserved regions in the alignment graph, targeted to amplify species- specific sequence entire F gene open reading frame (ORF) which is useful in molecular identification of baculovirus in unknown samples. So, the PCR product of SpliGV used to prepare a specific probe for the F gene of this type of virus. Results reflected that it is possible to infect S. littoralis larvae by PhopGV if injected into larval haemocoel, the resulted virus of this infection showed by using DNA hybridization technique to be encode to F gene homologous with the F gene of Spli GV, which is revealed that the resulted virus acquired this F gene sequence from the host genome after infection. Consequently, these results may infer that if genetic aberrations occur in the host genome, this may affect in baculoviral infectivity. So, this study aimed to investigate the effect of gamma radiation at doses around those used in Lepidopteran male sterilization technique, one of insect control program, on the susceptibility of the irradiated cells to virus infection. ACKNOWLEDGMENT My first of thanks and prayers to ALLAH for supporting me through all my life. Words can never express the feeling of gratitude and appreciation to all the people who gave me their precious help in this work. I wish to express my sincere appreciation and gratitude to my first teacher in my field of specialization; Dr. Omaima Khamiss Associate Prof. of Animal Cell Culture and Virology, Animal Biotechnology Dep., GEBRI, Minufiya University, for her continues supporting to me. A lot of thanks to all my supervisors for giving me the chance to working under their supervision. Special deep appreciation for Prof. Monem S. Bashandy and Dr. Adly M. Abd-Allah for their supporting to this work. Also, much thanks for my colleague Ahmed A. Raheem for his technical assistance. DEDICATION I dedicate this work to my parents, brothers, sisters and also to my wife for all the support they lovely offered during my study. LIST of ABBRIVIATIONS Type of budded virus protein in Autographa californica AC114 multinucleocapsid Nucleopolyhedroviruse Type of budded virus protein in Autographa californica AC124 multinucleocapsid Nucleopolyhedroviruse Type of budded virus protein in Autographa californica AC132 multinucleocapsid Nucleopolyhedroviruse Type of budded virus protein in Autographa californica AC58/59 multinucleocapsid Nucleopolyhedroviruse Type of budded virus protein in Autographa californica AC66 multinucleocapsid Nucleopolyhedroviruse Type of budded virus protein in Autographa californica AC73 multinucleocapsid Nucleopolyhedroviruse Type of budded virus protein in Autographa californica AC74 multinucleocapsid Nucleopolyhedroviruse Autographa californica multinucleocapsid AcMNPV Nucleopolyhedroviruse AoGV Adoxophyes orana Granulovirus AsGV Agrotis segetum Granulovirus BEVs baculovirus expression vectors Bm5 Cell line established from Bombyx mori ovarian tissue BmNPV Bombyx mori nucleopolyhedrovirus BTI-Tn- Cell line established from embryos of the cabbage looper 5B1–4 Trichoplusia ni BV budded virus cDNA Complementary strand of deoc\xy-ribo nuclic acid CFPV calyx fluid plus venom ClGV Cryptophlebia leucotreta Granulovirus CPVs cytoplasmic polyhedroses DNA Deoxyribo nucleic acid ECV extracellular virus efp Envelop fusion protein EGFP Envelop glyco- fusion protein EppoNPV Epiphyas postvittana nucleopolyhedrovirus ESEM Environmental scanning electron microscopy F gene Gene encoding for envelop fusion protein type F F protein Envelop fusion protein type GFP green fluorescent protein Type of glycoprotein act as envelop fusion protein in group I gp64 baculoviruses GV Granulovirus Gy Gray, radiation dose unit HaSNPV Helicoverpa armigera single nucleopolyhedrovirus Hi5 Cell line established from Trichoplusia ni HSV herpes simplex virus Hv-AM1 cell lines derived from pupal ovaries of Heliothis virescens Hz-AM1 cell lines derived from pupal ovaries of Helicoverpa zea Type of budded virus protein in Autographa californica IAP-2 multinucleocapsid Nucleopolyhedroviruse IPLB- Cell line established from pupal ovaries of the fall Sf21AE armyworm Spodoptera frugiperda JHE Juvenile hormone esterase LBAM lightbrown apple moth LC Lethal concentration envelope protein of insect retroviruses in the genus LD130 Errantivirus (family Metaviridae) LqhIT2 insect specific neurotoxin LT Lethal time MbPDV Microplitis bicoloratus polydnavirus MNPV Multicapsid Nucleopolyhedrovirus NOV non-occluded virus NPV Nucleopolyhedrovirus OB occlusion body ODV occlusion-derived virus Type of budded virus protein in Autographa californica ODV-E18 multinucleocapsid Nucleopolyhedroviruse occlusion-derived virus envelope protein gene in ODV-E56 Autographa californica multiple nucleopolyhedrovirus Type of budded virus protein in Autographa californica ODV-E56 multinucleocapsid Nucleopolyhedroviruse OpMNPV Orgyia pseudotsugata multiple nucleopolyhedrovirus OV occluded virion P10 One of baculoviral capsid proteins DNA binding protein in Autographa californica p6 multinucleocapsid Nucleopolyhedrovirus PDV polydnavirus PhopGV Phthorimaea operculella Granulovirus PMs peritrophic matrices Type of budded virus protein in Autographa californica PP31 multinucleocapsid Nucleopolyhedrovirus PTM Potato tuber moth PxGV Plutella xylostella Granulovirus REN restriction endonuclease analysis S2 Schneider 2 cells derived from Drosophila melanogaster Se301 Cell line established from Spodoptera exigua SFX Type of commercial available insect medium Sf21 Cell line established from Spodoptera frugiperda Sub clone cell line of IPLB-Sf21AE cells derived from Sf9 pupal ovaries of the fall armyworm Spodoptera frugiperda SlGV Spodoptera litura Granulovirus SlNPV Spodoptera litura Nucleopolyhedrovirus SNPV Single nucleocapsid Nucleopolyhedrovirus SpliGV Spodoptera littoralis Granulovirus SpliNPV Spodoptera littoralis Nucleopolyhedrovirus SpltMNPV Spodoptera litura multicapsid nucleopolyhedrovirus TCID50 50% tissue culture infectivity dose major capsid protein in Autographa californica VP39 multinucleocapsid Nucleopolyhedrovirus VSV-G vesicular stomatitis virus glycoprotein CONTENTS Table of Contents 1. INTRODUCTION ............................................................................. 1 2. Review of Literature ......................................................................... 5 2.1 Discovery of Baculoviruses ................................................................. 5
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