RESUMO
Thermostable DNA polymerase gene from Thermus aquaticus was cloned into constructed Taq from Thermus a Qaticus [pTTQ] plasmid using EcoRI and Sa/I sites with subsequent transformation in Escherichia coli strain [TOP10]. The use of Isopropyl-beta-D-thiogalactopyranosid [IPTG] as inducer of interested gene expression under control of the lac promoter was investigated. The optimization of enzyme induction by IPTG was determined at shake flask level to be 0.52mM at exponential growth phase. Enzyme preparation was performed by lysis the cultured cells. Afterwards, the cell suspension was incubated at 75°C to denature all heat sensitive proteins in the cell suspension that have been removed by subsequent centrifugation. Finally, the clarified supernatant containing heat resistant Taq DNA polymerase was collected and stored at -80°C. The activity of enzyme was compared with commercial Taq DNA polymerase, which remained when stored in buffer containing 50% glycerol, at -20°C. The purified enzyme had a molecular weight of 94 KDa, as estimated by SDS-PAGE and yielded appropriate enzyme activity comparing to the commercial Taq DNA polymerase
Assuntos
DNA Polimerase Dirigida por DNA , Thermus , Escherichia coli , Clonagem Molecular , Expressão GênicaRESUMO
Infectious bursal disease [IBD] is an economically important viral disease of chickens with worldwide distribution which suppresses the immune system of young chickens. VP2 is the major host-protective protein of infectious bursal disease virus [IBDV]. The encoding region of VP2 protein was PCR amplified from a plasmid containing a cDNA fragment of large genomic segment of IBDV, strain D78. This region of 1356 bp was inserted into a eukaryotic expression plasmid, pCDNA4, under the control of human cytomegalovirus [hCMV] immediate early enhancer and promoter. Plasmid DNA was transfected into COS-7 cell line and transient expression of VP2 from the constructed plasmid was characterized by dot blotting with a polyclonal antibody to IBDV