Construction Of HBV Targeted-Ribonuclease With Linker Inserted And Antivirus Activity

Hepatitis B virus (HBV) infection is one of major public health problems worldwide. And it can further cause cirrhosis and hepatocellular carcinoma. Although it can be prevented by mass vaccination, there are still 5% people showing no reaction.Current treatments are of limited efficacy. The sustained response rate of the approved high-dose interferon-a (IFN-a) therapy is about 30%. Nucleoside analogues such as lamivudine markedly reduces viral load but suffers from the emergence of drug resistance. Other approaches, such as immunotherapy and gene therapy are still in experimental level. The situation has spurred interests in alternative approaches to interfere with HBV replication.Based on the anti-virus strategy--CTVI, we designed a fusion proteinwhich comprises targeted molecule HBVc and effector hEDN (named as targeted-ribonuclease, TR), and have already successfully inhibited HBV replication in vitro. To enhance the inhibitory effect of HBV TR (named asTR-L) , a classic linker-(Gly4Ser)3 sequence has been inserted between HBVc and hEDN in order to keep the natural spacial structures of these two functional domains and reducing their spacial obstruction as well. Meanwhile, for the further use in small animals and potential clinical practice, we constructed TR-L using recombinant adenoviral vector, and determined its anti-HBV activity in vitro.Two oligo nucleotides were synthesized and annealed to form linker, and cloned into pcDNA3.1(-)/TR to produce pcDNA3.1(-)/HBc-linker. Then the hEDN fragment was PCR amplified and inserted into pcDNA3.1(-)/HBc-linker to form pcDNA3.1(-)/TR-L in which the effector and the target molecule were separated by a linker sequence. pcDNA3.1(-)/TR-L expression was identified by IFA. Solid phase RIA shows: there was a significant decrease of supernatant HBsAg and HBeAg concentration from pcDNA3.1(-)/TR-L compared to other negative controll groups (P<0.01), no significant decrease among negative controll groups(P>0.05); a decrease between pcDNA3.1(-)/TR-L and pcDNA3.1(-)/TR was observed (P<0.05);compared to mock transfective group , the concentration supernatant HBsAg and HBeAg was decreased by about 51.9% 61.4% respectively. Supernatant HBV-DNA content determined by fluorescent quantification PCR shows: there was a significant decrease from pcDNA3.1(-)/TR-L compared to other negative controll groups (P>0.01), no significant decrease among negative controll groups (P>0.05); a decrease between pcDNA3.1(-)/TR-L and pcDNA3.1(-)/TR was observed (P<0.05); compared to mock transfect group, the content of supernatant HBV-DNA declined by about 49.9% . MTT results suggested there were no significant differences among groups.In order to further use TR-L in vivo to evaluate its function, we use RAd as a substitutional vector.First shuttle plasmid with targeted fragments were constructed, by inserting fragments TR-L TR TRmut HBVc hEDN into plasmid pDC316 Then RAds were packaged identified, amplified and determined titers in 293 cells. The acquired titer of RAds is (107pfu/ml): TR-L: 4.1; TR: 4.5; TRmut:3.7; hEDN: 5.6; HBVc: 6.3; RAd: 5.7 respectively. RAd/TR-L expression was identified by IFA. Solid phase RIA shows: there was a significant decrease of supernatant HBsAg and HBeAg concentration from TR-L group compared to other negative contrail groups (P<0.01), no significant decrease among negative controll groups(P>0.05); a decrease between TR-L and TR group was observed (P<0.05);compared to mock infection group, the concentration supernatant HBsAg and HBeAg declined by about 65.3% , 62.7% respectively. Supernatant HBV-DNA content determined by fluorescent quantification PCR shows: there was a significant decrease from TR-L group compared to other negative controll groups (P<0.01), no significant decrease among negative controll groups(P>0.05); a decrease between TR-L and TR group was observed (P<0.05);compared to mock infection group, the supernatant HBV-DNA content of TR-L group declined by about 59.9% . MTT results suggested there were no significant differences among groups...