The Target Inhibitory Effect Of DHAV IRES Element By DNAzyme

In order to find a practicable targeted from the gene level for curing DHAV, we designed and synthesized six DNAzyme (DZ369, DZ454, DZ514, DZ454-7, DZ454-9 and DZ000) according to the sequence of DHAV IRES element, and then conducted a series of research about the cleavage to IRES and the inhibition to DHAV replication by DNAzyme, obtained the following results.As the pGEM-T/IRES for the template we got the RNA product of IRES structural element (DHAV-RNA) used the T7 transcription kit, and the transcript was about 361nt. Then mixed the DHAV-RNA and DNAzyme and with the Mg2+ participate in, and tested in 5.0% agarose gel electrophoresis. Results shown that those DZ369, DZ454 and DZ514 which has the complete enzyme active center and with the flanks can complementary pairing with substrate, completely, can cleavage the target RNA at the G-C or A-C locus, but the irrelevant control group DZ000 which has the complete enzyme active center but without the cutting substrates recognition motif cannot cleavage the target RNA. indicated that there was a strong specificity when DNAzyme cleavage the IRES.The DNAzyme can play a better role when the divalent metal ion participation in, and it had a difference cleavage activity when the different divalent metal ion took part in. The Mg2+ showed the highest acceleration to the activity of DNAzyme, the rest was in turn Zn2+、Mn2+、Ni2+ and finally was Co2+We cloned the target gene which include IRES structural element and Red gene into the pEGFP-Nl vector to get the recombinant plasmid pEGFP-Red-IRES-N1. And then the DEF were transfected with bicistronic vector, successful expressed the Red and EGFP fluorescent protein. But the expression of green fluorescence was only reach 60-65% compared with the control group, and it required more time. These data indicated that the IRES can start the gene expression which locted it’s downstream but the activity of IRES was weaker than CMV.Next the DEF were co-transfected with pEGFP-Red-IRES-N1 vector and DNAzyme, the result showed that each DNAzyme group can inhibite the expression of EGFP, and the inhibition of IRES-dependent translation achieved a peak when targeting position 454 with 10μmol/L DZs, changing the length of DZs flanks had no effect to its activity. Furthermore, there was no significant difference in the expression of red fluorescence between the recombinant plasmids and the control group, indicated that the initial protein of IRES-dependent was relativiely independence.Another important result was that the replication inhibition of DHAV-1 by DZ454, cellular debris was collected at different timing, and then detected the expression of 3D gene using one-step RT-PCR (FQRT-PCR), showed that the replication of virus reach the peak at 60h and then remain unchanged, and the suppressing effects on the replication of DHAV-1 was caused by DZ454 through the cleavage to IRES in the virus replication, and the maximum suppression occurred at 72h (10.38%) and then did not change.In conclusion, DNAzyme can cleavage the specific site of DHAV IRES in the participation of divalent metal ions, also showed some inhibition to the replicaton of DHAV, those results provide datas for future study the treatment of DHAV.