Inhibiting The Expression Of UL49 Gene And The Viral Replication Of HCMV By EGS

By a sensitive RT-PCR method,the mRNA of UL49 gene was detected in the human embryonic lung fibroblast(HELF) infected with the HCMV strain(AD169). It is so far the first evidence that the UL49 gene can be transcribed during the replication cycle of HCMV in the infected HELFs. Furthermore, the UL49 mRNA was also examined in the total RNA extracts from the HELFs that had been infected for different time. The results showed that in 1 hour,5 hours, 9 hours or 12 hours of postinfection the RT-PCR amplification for HCMV UL49 mRNA was negative,While in 24 hours, 48 hours and 72 hours of postinfection, the amplication were both positive. It suggested that the UL49 gene of HCMV might not be an immediate early gene, but possibly an early gene(P2).Aimed to the UL49 gene of HCMV, eight EGSs had been designed with the aid of computer as an library for screening. 1) Primarily screening in vitro. An in vitro screening system was firstly established by using an crude extract of RNase P from HeLa cells. Through this screening system, some EGSs were confirmed to be effective for the guiding cleavage to the 32P-labelled substrate. The effective EGSs were EGS1 ,EGS3, EGS4, EGS5, EGS6 and EGS7, respectively. Further comparing the guiding activities of the six effective EGSs, it was found that four of them including EGS1, EGS3, EGS6 and EGS7 possessed an strong activity, and the guiding cleavage rates of them were both more than 50%. While the other two EGSs(EGS4 and EGS5) possessed an relatively weak activities, the guiding cleavage rates of them were only 35% and 36% respectively. 2) screening in vivo. The four EGSs with strong activities in vitro were further investigated by an in vivo screening system. Firstly, a fusion expression vector was constructed by inserting the full length DNA fragment of UL49 gene to the 5'terminus of the GFP gene (green fluorescent protein) in the pGEFP plasmid. Secondly, cotransfection of the HeLa cell...