Inhibition of truncated region of HCMV IE2 gene expression by vector-based RNAi
Objective To study the inhibited effect of siRNA on the expression of truncated region of HCMV IE2 gene by vector-based RNAi technology. Methods After designing two siRNA sequences R1 and R2 based truncated region of HCMV IE2 gene, siRNA expression fragments containing U6 promoter were amplified from pLL3.7 by semi-nested PCR, siRNA expression vectors as effector plasmid pMD-T-shRNAR1 and pMD-T-shRNAR2 were constructed by cloning siRNA expression fragments into pMD-T simple vector. Meanwhile, the truncated region of HCMV IE2 gene was obtained from HCMV mRNA by RT-PCR. Eukaryotic expression vector pEGFP-C1-IE2 as target plasmid was constructed by cloning truncated region of HCMV IE2 into pEGFP-C1 on special direction. After effector and target plasmids were transfected into Hep-2 cell by different ratio by lipofectin mediation, effect of RNAi could be observed at different time points by fluorescence microscope. Results Three recombinant vectors were constructed by test-based PCR, restriction digestion and gene sequencing methods. Two obtained shRNA expression vectors were available. One which interference effect was about 100% could stop expression of truncated region of HCMV IE2 gene from 48 h to 72 h. When effector and target plasmids ration was 1:1 and 1:2, the other which interference effect was 98.4%-99% could obviously inhibit expression of truncated region of HCMV IE2 gene in the same time. Conclusion Expression of HCMV IE2 gene can be effectively inhibited by vector-based RNAi technology. This will offer a kind of study path in following: what is molecular action of HCMV IE2 gene in HCMV infection and proliferation? Can HCMV IE2 gene be accepted as an object of medicine study?