表达马传染性贫血病毒受体和荧光素酶报告基因的293细胞系的建立

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

摘要:

为了在体外精确、简便地测定马传染性贫血病毒(EIAV)的中和抗体和研究不同毒株与受体的亲和性,克隆了马慢病毒受体1(ELR1)cDNA并插入真核表达载体pcDNA3.1(+),构建了表达载体pELR1。该载体瞬时转染293细胞后,经Western blot和间接免疫荧光(IFA)检测,确认了ELR1的表达。在pELR1质粒的基础上,插入EIAV疫苗株前病毒基因组转录调控区LTR以及萤火虫荧光素酶报告基因(Luc)构建了表达载体pELR1-LTR-Luc,并转染293细胞,建立了ELR1-LTR-Luc(293-E)细胞系。该细胞系能稳定表达ELR1基因,并且能在LTR的调控下表达萤火虫荧光素酶基因。用1000TCID₅₀的EIAV驴胎皮肤细胞疫苗株D18V13接种该细胞,24h后检测其荧光素酶活性是未接毒对照的3.15倍。同时用IFA检测证明了病毒在细胞内的增殖。EIAV强毒株L21的接毒试验显示,ELR1-LTR(293-E)细胞的萤火虫荧光素酶活性与该毒株的接毒量在10^-2~10^-7稀释范围内呈正相关。该细胞系传35代后,外源基因的表达特征未发生改变。该细胞系的建立为进一步开展EIAV与细胞受体相互作用以及中和抗体评价等研究奠定的重要基础。

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.