Construction of Human Cytomegalovirus Han?ΔUS12?BAC Strain and Studies on Its Replication in Human Embryonic Lung Fibroblasts / 中国医科大学学报

ABSTRACT

Objective To construct human cytomegalovirus(HCMV)Han?ΔUS12?BAC strain and to study the role of US12 in HCMV replica?tion in human embryonic lung fibroblasts(HELF). Methods Kanamycin?resistant gene was amplified with primers containing homology arms se?quence flanking US12 and then electroporated into E.coli DY380?Han?wt?BAC competent cells. Successfully recombinant Han?ΔUS12?BAC clones were identified by PCR,sequenced for confirmation Han?ΔUS12?BAC plasmids were electroporated into HELF to produce infectious viri?ons. Han?ΔUS12?BAC and Han?wt?BAC were inoculated onto HELF at the multiplicity of infection of 0.1 pfu/cell. The viral titer in the supernatant was measured by TCID50 assay and growth kinetics of the viruses in HELF was studied. Results Han?ΔUS12?BAC clone was successfully con?structed. Han?ΔUS12?BAC was reconstructed in HELF to generate infectious virions. Growth kinetics assay indicated that Han?ΔUS12?BAC and Han?wt?BAC showed no differences in their growth and dissemination in HELF. Conclusion US12 in HCMV clinical strain Han is dispensable for HCMV growth and dissemination in HELF.