Expression of VP1 and VP2 capsid proteins from the Encephalomyocarditis virus isolated from nonhuman primates in Singapore

• Main Author: Chua, Eliza Hui-Ju
• Other Authors: Richard J Sugrue
• Format: Final Year Project
• Language: English
• Published: 2014
• Subjects: DRNTU::Science::Biological sciences::Molecular biology
• Online Access: http://hdl.handle.net/10356/61084
• Institution: Nanyang Technological University

The Encephalomyocarditis virus (EMCV) isolated from the tissue homogenate of the hearts of two deceased juvenile orang utans from the Singapore Zoological Gardens in Vero cell cultures is a highly divergent variant of EMCV. This variant resulted in the sudden deaths of four orang utans. The lowest sequence relationships compared to other EMCV strains was found in the variable VP1 capsid region, with the nucleotide sequence identities for the other capsid proteins also all falling below 80%. The nucleotide sequences of VP1 and VP2 capsid proteins for this EMCV heart isolate, designated Sing-M105-02, was obtained from NCBI, and both capsid protein sequences were ligated with the vector pRSET C then transformed into chemically competent EZ E.coli bacteria cells to be cloned. VP1 and VP2 were then retransformed into BL21(DE3)pLysS cells and protein expression induced using IPTG. VP1 and VP2 capsid proteins were found to be insoluble and were able to be purified by denaturing the proteins using 6M Guanidine HCl followed by binding to cobalt resin then eluting using imidazole.