الفهرس 
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Abstract
Equine herpesviruses, considered one of the most viral diseases affect not only equine and donkeys but also different species of amphibians, reptiles, dears, and cattle. Causing great economic losses, through respiratory disease, abortion, and may extended to nervous manifestations.
Glycoproteins either structural or non structural play an important role in the viral life cycle including attachment to the specific cells, penetration, replication, and egress from infected cells .Which have greet impact on the virulence of the virus. One of these glycoproteins was tegument protein encoded by EUL45 gene. For characterization of such gene, the following study was performed.
1- Construction of the UL45 EHV-1Ab4p gene (ORF15) prokaryotic expression plasmid, through recombination of the EUL45 N terminus with multiple cloning site of the pGEX-6P-1 plasmid, which transformed into E.Coli BL21. For induction of protein expression, different concentrations of IPTG (0.1, and 0.5 mM), different incubation times (over night, 3 h, and 5 h) with different incubation temperatures (20, 23, 25, 30, and 37°C) were applied, revealing that EUL45 exhibit high degree of expression under the following condition, 0.1 mM IPTG for 5 h at 37°C .
The purified EUL45 GST recombinant protein was assessed by SDS-PAGE analysis yielding high degree of purity indicated by one clear band at the predicted molecular size of 33.5 KDa .
The majority of EUL45 GST recombinant protein was soluble and present in the supernatant of purified samples.
2-The purified expressed pGEX-6P-1 N UL45protein was used for production of polyclonal antibodies in guinea pigs. Specificity and reactivity of the prepared polyclonal anti bodies was assessed by western blotting revealing that, the prepared anti bodies was specifically reacted with the EUL45 protein in the wild type Ab4p infected FHK cells, indicated by appearance of more than one specific band with a molecular weight ranging from 32-60KDa comparable to 33.5 KDa with the purified EUL45 GST recombinant protein. UL45-specific bands recognized by the UL45-specific antibody were of a higher molecular mass, suggesting that the respective UL45 proteins was posttranslationally modified to a higher-molecular-mass protein by glycosylation or phosphorylation as predicted by on line tools.
3- Intra cellular localization of EUL45 for wild type Ab4p virus was investigated through IFA on theAb4p infected RK13 cells, revealed the intra cytoplasmic localization of EUL45.
4-Time lapse of EUL45 protein expression with in the Ab4p infected RK13 cells assessed by IFA, revealed that 8 h post infection was the exact time for protein expression and that expression increased with the time.
5- In order to investigate the effect of recombination of EUL45 with a marker gene on the expression of such gene. Infusion cloning of EUL45 with pEGFP-C1 plasmid was constructed and IFA was done on transfected RK13 cells with the recombinant plasmid, revealed that pEGFP-C1 as expression plasmid had great influence on the EUL45 protein expression as it make the expression more early at 4 h post transfection but not affect the intra cytoplasmic localization of the protein. Also the protein was found to be associated with the Golgi apparatus starting from the expression time of UL45 at 8 h pi when using Golgi 58 K as a marker for Golgi apparatus.
6-Transcription initiation site for EUL45 was detected to be at nucleotide position of 21211 in Ab4p genome. Also it was found that a putative TATA box for UL45 gene Ab4p strain at nucleotide position of 21232-21237 by 5-SMARter RACE PCR.
7-EUL45 was one of the late viral proteins that present late during infection, it is expression was highly dependent on viral DNA synthesis indicated by absence of specific band of EUL45 in the PCR reaction preformed on RNA extracted from Ab4p infected FHK cells with 0.5 and 1mg /ml but not at 0.1 mg/ml phosphonoacetic acid as viral DNA synthesis inhibitor.