Research Information System
7th International Entomopathogens and Microbial Control Congress, Kayseri, Turkey, 11 - 13 October 2014, pp.1
Amsacta moorei
entomopoxvirus (AMEV) is an invertebrate virus that belongs to B type
entomopoxviruses. AMEV has potential to be utilized as gene expression vector,
gene therapy vector and biocontrol agent
against noxious insects. For
efficient use of AMEV for these purposes, the functions of produced proteins need
to be determined. The AMEV can be easily replicated and modified in insect cell
lines. This feature makes it suitable for virology studies. AMEV genome
includes 294 open reading frames. One of these ORFs is amv063. Bioinformatics
studies shown that AMV063 protein may be a Caspase-2 and may exhibit apoptotic
features. The aim of this study is the production of Amsacta moorei
entomopoxvirus AMV063 recombinant protein in prokaryotic expression system that
will be used in subsequent caspase assay experiments. For this purpose, amv063
gene was amplified with PCR by suitably designed primers. The amplified amv063
gene was cloned into the transfer vector (pGEM-T easy) and the sequence
was confirmed by Sanger sequencing as commercial. The amv063 gene in the
transfer vector was cloned into the expression vector (pET-28A) by Kan+
antibiotic selection. The AMV063 protein was expressed in E.coli BL-21
expression strain with IPTG induction. Expressed AMV063 protein was purificated
with His-Taq protein purification system and verified by western
blotting. To investigate the effect of AMV063
protein on apoptosis, the obtained protein will be tested on the sensitive CF-70-B2
cell line. Cells treated with AMV063 will be subjected to caspase assays to
investigate apoptotic responses in susceptible cells