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Construction and Cloning of Reporter-Tagged Replicon cDNA for an In Vitro Replication Study of Murine Norovirus-1 (MNV-1).
- Source :
-
Malaysian Journal of Medical Sciences . Nov/Dec2017, Vol. 24 Issue 6, p29-38. 10p. - Publication Year :
- 2017
-
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 NanoLucâ„¢ Luciferase gene was used to develop a reporter-tagged replicon system to study norovirus replication. Methods: The NanoLucâ„¢ 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: NanoLucâ„¢ 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. [ABSTRACT FROM AUTHOR]
Details
- Language :
- English
- ISSN :
- 1394195X
- Volume :
- 24
- Issue :
- 6
- Database :
- Academic Search Index
- Journal :
- Malaysian Journal of Medical Sciences
- Publication Type :
- Academic Journal
- Accession number :
- 130338733
- Full Text :
- https://doi.org/10.21315/mjms2017.24.6.4