Establishment of an MRC-5 based indicator cell line for detection of human cytomegalovirus

Objective To develop an indicator cell line for detection of human cytomegalovirus (HCMV) based on inducible expression of green fluorescent protein (GFP) gene. Methods GFP reporter gene and the promoter of HCMV UL54 gene (UL54p) were amplified by PCR method; GFP gene was used to replace the luc2 coding sequence in pGL4.17[luc2/Neo] vector, and then UL54p was inserted into the multiple cloning sites (MCS) to generate pGL4UL54p-GFP. pGL4UL54p-GFP plasmid was transfected into MRC-5T, a telomerase reverse transcriptase (TERT) gene-immortalized MRC-5 cell line, by mixing with lipofectamine 3000 reagent. G418-resistant cell colonies were isolated and subjected into screening by observing the inducible expression of GFP under fluorescence microscope after HCMV infection. The specificity to HCMV of the obtained cell line was tested through infection of human adenovirus type 5 or influenza A virus subtype H1N1. Results The recombinant plasmid pGL4UL54p-GFP was successfully constructed. Nine G418-resistant cell colonies were obtained, and two of them could express GFP after HCMV infection among which MRC-5TUG#7 gave brighter fluorescence. No GFP-positive cells were seen after challenging MRC-5TUG#7 with adenovirus or influenza virus, suggesting an acceptable specificity. Conclusion MRC-5-based indicator cell line for HCMV detection was successfully established, which may be used for HCMV isolation and titration. Key words: Cytomegalovirus; Cell, cultured; Gene expression; fluorescent antibody technique