Construction of HBV-specific ribozyme and its recombinant with HDV and their cleavage activity in vitro

AIM To construct the recombinant of HDVcDNA and HBV-specific ribozyme gene byrecombinant PCR in order to use HDV as atransporting vector carrying HBV-specificribozyme into liver cells for inhibiting thereplication of HBV.METHODS We separately cloned the ribozyme（RZ）gene and recombinant DVRZ（comprisingHDV cDNA and HBV-specific ribozyme gene）intothe downstream of T7 promoter of pTAdv-Tvector and studied the in vitro cleavage activityof their transcripts（rRZ,rDVRZ）on target RNA（rBVCF）from in vitro transcription of HBV Cgene fragment（BVCF）.RESULTS Both the simple（rRZ）and therecombinant ribozyme rDVRZ could efficientlycatalyze the cleavage of target RNA（rBVCF）under different temperatures（37℃,42℃ and55℃）and Mg2+concentrations（10 mmol/L,15 mmol/L and 20 mmol/L）and their catalyticactivity tended to increase as the temperaturewas rising.But the activity of rRZ was evidentlyhigher than that of rDVRZ.CONCLUSION The recombinant of HDV cDNAand ribozyme gene had the potential of beingfurther explored and used in gene therapy of HBVinfection.

AIM:To construct the recombinant of HDV cDNA and HBV specific ribozyme gene by recombinant PCR in order to use HDV as a transporting vector carrying HBV-specific ribozyme into liver cells for inhibiting the replication of HBV.METHODS:We separately cloned the ribozyme (RZ) gene and recombinant DVRZ (comprising HDV cDNA and HBV-specific ribozyme gene) into the downstream of T7 promoter of pTAdv-T vector and studied the in vitro cleavage activity of their transcripts (rRZ, rDVRZ) on target RNA (rBVCF) from in vitro transcription of HBV C gene fragment(BVCF).RESULTS:Both the simple (rRZ) and the recombinant ribozyme rDVRZ could efficiently catalyze the cleavage of target RNA (rBVCF) under different temperatures (37 degrees , 42 degrees and 55 degrees ) and Mg(2+) concentrations (10mmol/L, 15mmol/L and 20mmol/L) and their catalytic activity tended to increase as the temperature was rising. But the activity of rRZ was evidently higher than that of rDVRZ.CONCLUSION:The recombinant of HDV cDNA and ribozyme gene had the potential of being further explored and used in gene therapy of HBV infection.