Construction of a eukaryotic expression vector harboring the small interfering RNA targeting HCMV-IE1 gene and its gene silencing efficiency / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effect of RNA interference targeting human cytomegalovirus immediate early gene 1 (HCMV- IE1) on the gene expression in vitro.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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).</p><p><b>CONCLUSION</b>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.</p>

Proteomic study of the brain tissues of mice with human cytomegalovirus infection / 南方医科大学学报

<p><b>OBJECTIVE</b>To establish the two-dimensional electrophoresis profiles with high resolution and reproducibility from the brain tissues of mice with human cytomegalovirus (HCMV) infection.</p><p><b>METHODS</b>Forty Kunming mice were randomized into HCMV infection group (n=20) with HCMVAD(169) injection and control group (n=20) with saline injection in the brain. Thirty days after the injections, the brain tissue of the mice were taken and the protein fractions were isolated by two-dimensional gel electrophoresis (2-DE). Image Master 2D software was used to identify the differentially expressed proteins, and the peptide mass fingerprint (PMF) data were obtained for identification of the differential protein spots via database searching. Western blotting was performed to verify the expressions of some of the differential proteins.</p><p><b>RESULTS AND CONCLUSION</b>The 2-D maps of the brain tissues with high Well resolution and reproducibility were obtained. Some of the differentially expressed proteins identified by mass spectrometry (MS) matched their counterparts in the SWISS-2DPAGE database. Western blotting analyses verified the differential expression of the individual proteins. These data can be of value for studying the diagnosis, pathogenesis and effective therapeutic targets of the disease.</p>