Establishment of a 293-cell line containing luciferase reporter for EIAV receptor and LTR functions

Abstract:

To accurately and conveniently detect neutralizing antibodies and receptor binding affinities of different equine infectious anemia virus (EIAV) strains, the cDNA of EIAV receptor, ELR1, was cloned and inserted in an eukaryotic expression vector pcDNA3.1(+). This recombinant plasmid was designated as pELR1. The 293 cell line was transiently transfected with pELR1 and the expression of ELR1 on transfected cells was verified by Western blot and indirect immunofluorescence assay (IFA). Furthermore, the transcription regulatory region, long terminal repeat (LTR), of an EIAV vaccine strain and a reporter of firefly luciferase gene were tandemly cloned into pELR1. The resultant expression vector, which was designated as pELR1-LTR-Luc, was used to transfect 293 cells. A transfected cell line, ELR1-LTR-Luc (293E) which consistently expressed ELR1 and produced luciferase under the regulation of LTR, was isolated and further characterized. The entrance and replication of EIAV in ELR1-LTR-Luc (293E) cells were verified by IFA. The luciferase activity in the cell line treated with 1000 TCID50 of an EIAV vaccine strain for 24h was increased by 2.15 folds when compared with the activity in untreated cells. Furthermore, the luciferase activities in the cell line were linearly correlated with the doses of inoculated EIAV virulent stain L21 diluted at 10^-2～10^-7. The transfected genes in the ELR1-LTR-Luc (293E) cell line were consistently expressed during 35 passages of the host cells. This ELR1-LTR-Luc report system can be used for the study of interaction between EIAV strains and the receptor, as well as for the evaluation of neutralizing antibodies raised by EIAV.