Cloning of Viral Proteases Lb and 3C of FMDV and Expression in BHK-21 Cells

Saturday, February 13, 2016
Dulcemaria Hernandez, University of California, Irvine, Irvine, CA
Foot-and-mouth disease virus (FMDV) is a highly contagious picornavirus among important livestock species such as cattle and swine. The genome of FMDV is composed of a positive-strand RNA that codes for a single polyprotein that is cleaved by two viral-encoded proteases, Lb and 3C. Both Lb and 3C are known to play important roles in evading the host innate antiviral response during infection. The purpose of this study is to clone viral proteases Lb and 3C to further investigate their role in the innate antiviral response. The sequences of both proteases were amplified using PCR from FMDV isolate O1Kb (serotype O) RNA. The amplified Lb and 3C gene fragments were ligated into vector pcDNA3.1 and the resulting plasmids, pLb-O1K and p3C-O1K, were transformed into competent DH5α cells. Colonies containing the plasmids were selected through their ampicillin resistance. Successfully transformed colonies were lysed and double digested with BamHI and XbaI in order to confirm that they contained the Lb or 3C gene fragments. Four colonies, two that cloned protease Lb and two that cloned protease 3C, were also sent for sequencing to determine gene integrity. BHK-21 cells were then transfected with plasmids pLb-O1K and p3C-O1K. After 24 hours, the cells were lysed and analyzed for protein expression through SDS-PAGE and Western blot. Protease Lb was detected, however, there was no detection of protease 3C. New experiments performed in Madrid have recently confirmed expression of protease 3C. The constructed plasmids are being used for further studies to better understand the importance of proteases Lb and 3C in viral survival by avoiding the host innate antiviral response and provide insight for possible antiviral targets.