Inhibition Of Gene Expression Of HCMV UL49 By Intracellular Expression Of External Guide Sequence RNA

RNase P was a ribonucleoprotein complex, which was responsible for the 5' maturation of precurosor tRNAs, and abroad existed in Archaebacteria, bacterium, yeast and animals. RNase P could be directed to cleave any RNA when the target mRNA formed a complex with a short, complementary oligonucleotide called external guide sequence (EGS), and at the same time, the stucture of the complex was similar to that of precurosor tRNA.Now the function of UL49 is still not being figured out. Some researcher considered that it encoded the capsid protein of human cytomegalovirus(HCMV), according to its coding sequence, and played a significant role in the replication and multiplication of virus.First of all, six EGSs was designed on the similar principle of Wen-jun Zhang 's (EGS6 was a positive control), based on the results of cleavage experiments targeting UL49 in vitro which were done by Zhang, then they were constructed into the eukaryotic expression vector pcDNA3.1-, for studing whether they could effectively guide RNase P in viro to cleavage UL49 gene segment, which were cloned into the vector pEGFP-N1 and named UI49-A-GFP. To achieve this aim, some experiments have been done over the last two years.After that, six EGSs recombinant plasmid were separately co-transfected with UI49-A-GFP into Hela cells. By analysing the change of green fluorescence with Typhoon, an instrument which could be used to scan fluorescence, we got preliminary results and assessed the dosage in the next experiments. Furthermore, we studied the functions of EGSs by flow cytometer, real-time PCR. The results showed that five EGSs all had marked guiding cleavage activity, and the effect of pDU6-EGS2 was the most significant in the five EGSs.In sum, a series of experiments was designed to test whether EGSs which were active in vitro were still effective in vivo, based on the experiments in vitro. And then the effective EGSs, which were selected by the experiments in vivo, would be used further research of UL49 gene and the next virus experiments. By the analysis of essay data, we were able to...