Construction of a eukaryotic expression vector harboring the small interfering RNA targeting HCMV-IE1 gene and its gene silencing efficiency.
- Author:
Ni XIE
1
;
Yan-ping HAN
;
Jin-bin WU
;
Jian-hui YUAN
Author Information
- Publication Type:Journal Article
- MeSH: Antigens, Viral; biosynthesis; genetics; Cell Line; Genes, Immediate-Early; Genetic Vectors; genetics; Humans; Immediate-Early Proteins; biosynthesis; genetics; RNA Interference; RNA, Small Interfering; genetics; Recombinant Proteins; biosynthesis; genetics
- From: Journal of Southern Medical University 2010;30(9):2077-2079
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo investigate the effect of RNA interference targeting human cytomegalovirus immediate early gene 1 (HCMV- IE1) on the gene expression in vitro.
METHODSAccording to the sequence of HCMV-IE1 gene, the small interfering RNA (siRNA) sequences were designed and introduced into the eukaryotic expression vector containing the U6 promoter. After verification by sequence analysis, the recombinant eukaryotic plasmid (pHCMV-IE1i) was transfected into HEL HCMVAD169 cells. The effectiveness of HCMV-IE1 gene silencing was investigated by fluorescence microscopy, flow cytometry and RT-PCR.
RESULTSSequence analysis confirmed successful construction of the recombinant eukaryotic expression plasmid pHCMV-IE1i. The expression of HCMV-IE1 was effectively suppressed by pHCMV-IE1i transfection in HEL cells as shown by fluorescence microscopy, flow cytometry (P < 0.05) and RT-PCR (P < 0.05).
CONCLUSIONThe expression of HCMV-IE1 can be effectively suppressed by RNA interference technique in vitro, which provides experimental data for prevention and treatment of HCMV infection.
