1. Construction and Cloning of Reporter-Tagged Replicon cDNA for an In Vitro Replication Study of Murine Norovirus-1 (MNV-1)
- Author
-
Muhammad Khairi Ahmad, Muhammad Yunus, Nurulisa Zulkiflie, Rafeezul Mohamed, Mowaffaq Adam Ahmed, Yasser M Tabana, Ida Shazrina Ismail, and Doblin Sandai
- Subjects
0301 basic medicine ,Reporter gene ,business.industry ,ved/biology ,viruses ,ved/biology.organism_classification_rank.species ,General Medicine ,Amplicon ,Molecular biology ,Fusion protein ,Fusion gene ,03 medical and health sciences ,030104 developmental biology ,Viral replication ,Complementary DNA ,Medicine ,Original Article ,Replicon ,business ,Murine norovirus - Abstract
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.
- Published
- 2017