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[Construction of a eukaryotic expression vector harboring the small interfering RNA targeting HCMV-IE1 gene and its
Ni Xie1, Yan-ping Han, Jin-bin Wu
1First Affiliated Hospital of Shenzhen University/ Second Shenzhen People's Hospital, Shenzhen 518035, China. gracexn2001@hotmail.com
Objective:
To investigate the effect of RNA interference targeting human cytomegalovirus immediate early gene 1 (HCMV- IE1) on the gene expression in vitro.
Methods:
According 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.
Results:
Sequence 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).
Conclusion:
The 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.
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