Yan Ma
ABSTRACT
EcoHIV/NDK is a recombinant virus modified from HIV-1 that uses mice as a host. NanoLuc (Nluc) luciferase is a genetically engineered small molecule enzyme (19.1kDa). It is currently the best bioluminescence reporter gene which uses furimazine as a luminescent substrate to produce detectable fluorescence. In order to quantify and locate the levels of EcoHIV/NDK infection and expression, the EcoHIV/NDK plasmid and the Nluc luciferase gene were integrated together to construct EcoHIV/NDK-NanoLuc virus. To verify whether EcoHIV/NDK-NanoLuc is effective in in vivo and in vitro, we conducted the following experiments. First, the existence of the nanoluc fragment in the recombinant plasmid was verified by PCR amplification, and then the correctness of the plasmid construction was verified by restriction enzyme digestion. As a result, the correct recombinant and plasmid were constructed. The Nluc luciferase reporter gene was used to indicate the infection of the successfully packaged virus on Hela-mCAT cells and the three strains of Kunming mice, BALB/c nude mice, and 129S2/SvPasCrl mice, respectively. Our results show that EcoHIV/NDK-NanoLuc can effectively infect Hela-mCAT cells and three strains of mice, in which viral replication site and distribution was detected through the luciferase reporter gene.
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