Cloning and expression of Pteropine Orthoreovirus sigma C protein in bacterial cells

Abstract

Introduction: Pteropine Orthoreovirus (PRV) was first cloned in 2011 to represent a collective of orthoreoviruses isolated from bat species of the genus Pteropus . Humans, especially infants or children, infected with Mammalian Orthoreovirus (MRV), a genetically closely related ‘ relative’ to PRV, commonly present with mild upper respiratory tract illness or enteritis. However, this does not hold true for PRV, as acute respiratory syndrome is commonly seen in patients infected with PRV. Objectives: The focus of this study was to clone the sigma C gene in two vectors (pMal-C5X and pMal-P5X) and express it in different bacterial cells (ER2523 and BL21). However, the targeted receptor will be identified in a future study. Materials and methods: The sigma C gene of PRV1K (Kampar virus), PRV2P (Pulau virus) and PRV3M (Melaka virus) were amplified and cloned into (pMal-C5X and pMal-P5X) vectors. Colony polymerase chain reaction (PCR) was preformed to screen for positive clones prior to being expressed in BL21 and ER2523 bacteria. Periplasmic and total lysis extraction was performed on both bacterial cells prior to separation by SDS-PAGE (sodium dodecyl sulphate polyacrylamide gel electrophoresis) and visualization with Coomassie Blue stain. Results: The PCR of sigma C gene for three different viruses, namely Pulau, Melaka and Kampar, were performed. PCR of Pulau and Kampar viruses were successful but Melaka virus was unsuccessful because it did not reach the desired band and was excluded. The protein receptors were confirmed by a restriction enzyme reaction. Conclusions: Sigma C of PRV2P was expressed in bacterial cells without any lethal effect on the bacteria. The protein will be purified and targeted receptor identified in a future study. Acknowledgements: We would like to acknowledge our University and our Dean, Dr Khalid Al Qumaizy, for giving us this great opportunity, and we acknowledge Dr Keeny Voon from International Medical University of Malaysia for helping us conduct this research.