Construction Eukaryotic Expression Vector For M1GS Targeting HCMV UL54 Gene And Detection The Activity Of Two M1GSs In Vivo

RNase P from E.coli is a ribonucleoprotein complex responsible for the 5' maturation of tRNAs. M1RNA, the catalytic RNA subunit of RNase P, can cleavage substrate in vitro and in vivo, even if C5 protein lacks. RNase P can be directed to cleave any RNA when the target mRNA is in a complex with a short, complementary oligonucleotide called an external guide sequence (EGS). The EGS can be covalently linked to Ml RNA, forming a new kind of ribozyme—M1GS. It has the cleavage function stronger than M1RNA and EGS.UL54 gene, encoding the DNA polymerase of HCMV, is a key for the virus duplication. Moreover, the mutation of UL54 can resist the drugs which treat HCMV. So it is necessary to interfere the function of the UL54 gene for HCMV treat.Based on the M1GS cleavage effect targeting UL54 in vitro by Master Chen Hao-jun, we selected two of seven specific cleavage M1GS, T4-M1GS and T7-M1GS, which were constructed into the retrovirus vector—pLXSN with U6 promoter, for further studying if they can cleavage UL54 gene segment, which were cloned into the vector pEGFP-N1, in vivo.T4-M1GS and T7-M1GS were cloned into the retrovirus vector pLXSN respectively, namely T4-M1GS-pLXSN and T7-M1GS-pLXSN.The two M1GS didn't interfere the reporter gene—GFP, detected by the M1GSs acting the vector pEGFP-N1 in Hela cell.T4-M1GS could cleavage UL54-C when they existed in the same cell through fluorescence scanning and Northern blot analysis. When T4-M1GS-pLXSN and UL54-C-GFP co transfected Hela cell, T4-M1GS cleavageed UL54-C partially. When UL54-CD-GFP transfected the cell which expressed T4-M1GS stablely, T4-M1GS could cleavage UL54-CD almost completely.T7-M1GS could also cleavage UL54-CD with Northern blot after they co transfected Hela cell.The research results indicated the two M1GSs also had high cleavage activity invivo and the experiment that MlGSs were screened in vitro was necessary. Because the human fibroblast cell is the unique host for HCMV in vitro and the human fibroblast is hard to be transfected except virus. We constructed MIGS into retrovirus vetor for later work. And we proved that MIGS was a useful genetool for anti-virus.