| OBJECTIVESHuman cytomegalovirus (HCMV) is a widespread human pathogen that causes serious consequences in different patients. RNA interference (RNAi) is a powerful and potent technique to treat HCMV infection. HCMV UL122 gene and UL54 gene are essential genes for HCMV replication and infection. The sufficient knowledge of effective RNAi target site in UL122 gene and UL54 gene is still in shortage. Therefore, it is of great importance to find the most effective RNAi target sites in HCMV UL122 gene and UL54 gene.MethodsThis study included four parts:(1) Construction of fusion protein expression vectors:A truncated cDNA fragment of UL122 gene and the full-length of cDNA of UL54 gene were amplified from the genomic cDNA of HCMV AD 169 strain and cloned into pEGFP-N1 to construct fusion protein expression recombinant plasmids pUL122-EGFP and pUL54-EGFP. These two vectors can express enhanced green fluorescence protein (EGFP) for detection. (2) Construction of small hairpin RNAs (shRNA) expression vectors:Target sites corresponding to the UL122 gene and UL54 gene were selected and cloned into plasmid pAVU6+27 to construct shRNA expression recombinant vectors.(3) Cotransfection of fusion protein expression vectors and shRNAs expression vectors:Fusion protein expression vectors and shRNAs expression vectors were contransfected into AD293 cells. The inhibition effects of the expression of fusion protein were detected by fluorescence microscope, flow cytometry (FCM) and fluorescence quantitative real-time PCR for the screening of the most effective RNAi target sites.(4) Construction of lentiviral vectors and contransfection:In order to explore the long term knock-down regulation of RNAi, we constructed lentiviral vectors according to the effective RNAi target sites screened out using plasmid vector. The lentiviral vectors were then cotransfected into 293T cells to eveluate its know-down effects by FCM and Western blot.Results(1) The fusion protein expression vectors pUL122-EGFP and pUL54-EGFP were successfully constructed. The fluorescence can be detected under fluorescence microscope 24 h and 48 h after transfection. The green fluorescence signals transfected after 48 h were stronger than the green fluorescence signals transfected after 24 h.(2) Six target sequences corresponding to the open reading frame of HCMV UL122 gene and UL54 gene were selected to construct siRNA expression vectors, which were designated as psiUL122-1, psiUL122-2, psiUL122-3, psiUL54-1, psiUL54-2 and psiUL54-3. (3) The shRNA expression vectors were then cotransfected into AD293 cells with fusion protein expression vectors. The difference of the expression of green fluorescence signals between different groups was not apparent 24 h post-cotransfection. However, the green fluorescence signals were significantly reduced 48 h post-cotransfection in the cells cotransfected with pUL122-EGFP and psiUL122-1, psiUL122-2 separately, whereas green fluorescence signal was slightly reduced in the cells cotransfected with pUL122-EGFP and psiUL122-3. The inhibitory effects of the siRNAs on the expression of EGFP were further quantitatively validated by FCM assay 48 h post-transfection. The EGFP expression of the cells transfected with psiUL122-1 and psiUL122-2 was decreased by 82.0%±1.0% and 79.5%±2.5%, respectively. However, psiUL122-3 resulted in a weak reduction in the EGFP expression with an inhibitory rate of 53.5%±2.5%. We then examined the knock-down effectiveness of siRNAs on mRNA level, which was analyzed by fluorescence quantitative real-time PCR at 48 h post-transfection. A remarkable reduction of the expression of pUL122-EGFP mRNA was produced by psiUL122-1 and psiUL122-2, with an inhibitory rate of 97.3%±0.6% and 98.0%±0.1%, respectively. And the inhibitory efficiency of psiUL122-3 was 90.0%±3.5%.(4) The fusion protein expression vector pUL54-EGFP was cotransfected with psiUL54-1, psiUL54-2 and psiUL54-3, respectively. The difference of the expression of green fluorescence signals between different groups was not apparent 24 h post-cotransfection. The green fluorescence signals were significantly reduced 48 h post-cotransfection in the cells cotransfected with pUL54-EGFP and psiUL54-1, whereas fluorescence signal was slightly reduced in the cells cotransfected with pUL54-EGFP and psiUL54-2, psiUL54-3 separately. When evaluated with FCM, the EGFP expression of the cells transfected with psiUL54-1 was decreased by 85.4%±1.2%. However, psiUL54-2 and psiUL54-3 resulted in a weak reduction in the EGFP expression with an inhibitory rate of 14.9%±2.9%和20.4%±6.2%, respectively. A significant reduction of pUL54-EGFP mRNA was produced by psiUL54-1 with an inhibitory rate of 97.4%±0.7%. And the inhibitory efficiency of psiUL54-2 and psiUL54-3 was 20.3%±6.9%and 28.2%±5.6%, respectively.(5) Lentiviral vectors were successfully constructed and contransfected into 293T cells with pUL54-EGFP. Lentiviral vector PscSI-1 was found to have a mild inhibition of pUL54-EGFP fluorescent signals 24 h after contransfection and the inhibition effects become remarkable 48 h and 72 h after contransfection under fluorescence microscope. However, PscSI-2,PscSI-3 and PscSI-4 showed no inhibition of pUL54-EGFP fluorescent signals. When PscSI-1 and pUL54-EGFP were cotransfected with 1:2 ratio and 1:1 ratio, Western blot analysis displayed that PscSI-1 have significant knock-down of pUL54-EGFP both at these two ratios. PscSI-3 had mild inhibition of pUL54-EGFP at the ratio of 1:1. PscSI-2 and PscSI-4 showed no inhibition of pUL54-EGFP at these two ratios.Conclusion1. Recombinant plasmids pUL122-EGFP and pUL54-EGFP can express fusion protein UL122-EGFP and UL54-EGFP. And the green fluorescence signals can be detected from these two fusion protein. The fusion protein expression is remarkable 48 h after transfection.2. The target sites 618-638bp (psiUL122-1) and 1103-1123bp (psiUL122-2) of UL122 gene are effective RNAi target sites. The target site 1414-1434bp (psiUL122-3) is not an effective RNAi target site.3. The target sites 1479-1497bp (psiUL54-1 and PscSI-1) of UL54 gene is an effective RNAi target site. The target sites 2419-2437bp (psiUL54-2), 2886-2904bp (psiUL54-3 and PscSI-2),3058-3076bp (PscSI-3) and 419-437bp (PscSI-4) are not effective RNAi target sites.4. Recombinant shRNA expression plasmid vectors have the most effective inhibition of fusion protein at the time point of 48 h after cotransfection and have no significant inhibition at the time point of 24 h after cotransfection.5. Lentiviral vector is a kind of vector that shows inhibition effect quickly and has a higher transfection rate, more efficacious and stable knock-down effect, compared with plasmid vector.6. Lentiviral vector shows good accordance with plasmid vector for the the screening of effective RNAi target site.
|
PDF Full Text Request