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ObjectiveTo study the UL148 gene expression of human cytomegalovirus (HCMV) clinical isolate inhibited by external guide sequences (EGS), for investigating the gene function of UL148 and searching a new treatment of HCMV disease.MethodsExtracellular experiment: 1. Based on the character of UL148 RNA sequence, DNA-EGS was designed and synthesized; 2. PCR was used to amplify the UL148 gene from HCMV clinical isolate genome, and the product was cloned to pGEM-T-EASY plasmid. UL148 small fragment was amplified by PCR. The product was used to generate a DNA-template for transcription in vitro. After adding 32P-UTP, the UL148 small fragment RNA was transcribed; 3. The M1RNA gene amplified from the genome of E.coli DH5α, was cloned to pUC18 plasmid. After linearization, the M1RNA was transcribed in vitro; 4.The UL148 RNA, EGS and M1RNA mixed in cleavage buffer at 37℃for 1 hour, then the products separated by polyacrylamide gel.Intracellular experiment: After UL148 gene cloning to pEGFP-N1 plasmid, the recombinant plasmid was transfected to HeLa cell, while the pEGFP-N1 plasmid was transfected as negative control. And they were screened by G418 (1mg/mL)for 4 weeks. We chose the EGS4, which has the effective cleavage in extracellular experiment, for intracelluar experiment. After screening of G418, EGS4 was transfected to the HeLa cells at different concentration of 50nM, 100nM, 200nM and 400nM. After 24 hours, we observed the flourescence quant by fluorescence microscope. And using quantititive real time PCR, we analyzed the UL148 expression level. ResultExtracellular experiment: 1.We designed 5 EGSs; 2.The pT148 recombinant plasmid was established successfully. The UL148 small fragment RNA labelling by 32P-UTP was procured in vitro after transcription; 3. We sucessfully constructed pUC-M1 recombinant plasmid. After linearization, the M1RNA was synthetized by T7 RNA polymerase; 4. After screening by extracellular experiment, we identified EGSs that have the capability of cleavage of UL148 RNA.Intracellular experiment: The p148-EGFP recombinant plasmid was established successfully. After transcription of p148-EGFP and pEGFP-N1 to HeLa cells and screening of G418, we successfully established the UL148-HeLa cells for stable expression of UL148 and the EGFP-HeLa cells for stable expression of EGFP. Under the process of EGS4, the fluorescence intensity of UL148-HeLa cells attenuated significantly, but this effect didn’t show in the EGFP-HeLa cells. The analysis of flourescence quantitation PCR show that when the concentration of EGS4 was 50nM, the inhibition efficiency was 74.9 %(P<0.05), and then 100nM was 78.6%(P<0.05), 200nM was 81.1%(P<0.05), and 400nM was 87.0%(P<0.05).Conclusion1. After screening of extracellular experiment, we identified EGS1 and EGS4 have the capability of cleavage of UL148 RNA;2. The inhibition effects of EGS4 on UL148 expression was concentration- dependent;3. The extracellular cleavage system can develop to a screening pathway for intrecellular system;4. The EGS after screening of extracellular and intrecellular experiments, can use for study on UL148 gene function, and develop to a new micromolecule drug for anti-HCMV.
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