Malaysian Journal of Medical Sciences
School of Medical Sciences, Universiti Sains Malaysia
Vol. 24, No. 6, 2017, pp. 29-38
Bioline Code: mj17076
Full paper language: English
Document type: Research Article
Document available free of charge
Malaysian Journal of Medical Sciences, Vol. 24, No. 6, 2017, pp. 29-38
© Copyright 2017 - Penerbit Universiti Sains Malaysia
Construction and Cloning of Reporter-Tagged Replicon cDNA for an In Vitro Replication Study of Murine Norovirus-1 (MNV-1)|
Muhammad Khairi, Ahmad; Yasser, M Tabana; Mowaffaq, Adam Ahmed; Doblin, Anak Sandai; Rafeezul, Mohamed; Ida Shazrina, Ismail; Nurulisa, Zulkiflie & Muhammad Amir, Yunus
Background: A norovirus maintains its viability, infectivity and virulence by its ability to replicate. However, the biological mechanisms of the process remain to be explored. In this work, the NanoLuc™ Luciferase gene was used to develop a reporter-tagged replicon system to study norovirus replication.
Methods: The NanoLuc™ Luciferase reporter protein was engineered to be expressed as a fusion protein for MNV-1 minor capsid protein, VP2. The foot-and-mouth disease virus 2A (FMDV2A) sequence was inserted between the 3’end of the reporter gene and the VP2 start sequence to allow co-translational ‘cleavage’ of fusion proteins during intracellular transcript expression. Amplification of the fusion gene was performed using a series of standard and overlapping polymerase chain reactions. The resulting amplicon was then cloned into three readily available backbones of MNV-1 cDNA clones.
Results: Restriction enzyme analysis indicated that the NanoLucTM Luciferase gene was successfully inserted into the parental MNV-1 cDNA clone. The insertion was further confirmed by using DNA sequencing.
Conclusion: NanoLuc™ Luciferase-tagged MNV-1 cDNA clones were successfully engineered. Such clones can be exploited to develop robust experimental assays for in vitro assessments of viral RNA replication.
luciferases; foot-and-mouth disease virus; norovirus; capsid proteins; virus replication; replicon; restriction mapping
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