Effects Of Replication-Defective HBV Recombinant On Wild-Type Virus

Despite efficient vaccines for prophylaxis and numerous antiviral agents such as interferon-alfa(IFN-α) and nucleos(t)ide analogues,chronic hepatitis B virus(HBV) infection remains to be a major public health problem,which involves more than 350 million people in the worldwide.Recent years has witnessed great progress in the technology against HBV by gene therapy,such as ribozyme,dominant negative mutant(DNM) and small interference RNA(siRNA).Efficient and targeted introduction of theoretical gene and its permanent existence are crucial to successful gene therapy.In this research,a bi-expressive and replication-defective HBV recombinant was constructed based on the highly-efficient adenovirus vector,which contained a DNM composed of package signal(epsilon),C-S fused gene(C-S) and untranslatable X gene(X^-) of HBV,and the interleukin-2(IL-2) gene linked to HBV DNM by internal ribozyme entry site(IRES).Introduction of the recombinant into hepatocytes infected with HBV was anticipated to efficiently inhibit the replication of HBV by competitively depriving wild-typed HBV of C and S proteins and antiviral effect of IL-2.Meanwhile,unfavorable effect arising from the trans-activation of X-gene was minimized by product of X^-.Part one Construction of Replication-Defective HBV Recombinant Based on Adenovirus VectorTwo HBV gene fragments(2.4kb and 1.8kb) were acquired separately by treating the parental plasmid contaning 1.3-fold HBV genome with Kpnâ… /Xbaâ… and Xbaâ… / Hindâ…¢,and were transferred to the pUC19 vector independently to construct new recombinants 2.4Kb/pUC19 and1.8Kb/pUC19.The genetic code of the 8^th amino of X gene in both recombinants was mutated from CAA to terminal code TAA with a site-directed mutagenesis kit.With the same kit,the terminal code of C gene in the mutated 2.4Kb/pUC19 was replaced with the target sequence of Mluâ… (-ACGCGT-), and a new target sequence of Mluâ… was inserted before the origin code of S gene.The re-mutated 2.4Kb/pUC19 was treated with Mluâ… to remove fragment between C gene and S gene and result in fused C-S gene,followed by removing site of Mluâ… .The gene fragment C-S+X^- was then transferred to the adenovirus vector PDC316 and resulted in replication-defective HBV recombinant.Part two Construction of Bi-expressive Recombinant CS+X^--IRES-â…¡-2/PDC316Target sequence of restricted endonuclease Salâ… was added to the two terminals of IRES gene by PCR with plasmid IRES/pMD18-T as amplification template,and then IRES gene with Salâ… sites was cloned into pMD18-T.After confirmation by RFLP and sequencing,IRES gene was directionally transferred to adenovirus vector PDC316.IL-2 gene was acquired from plasmid IL-2/MNSM and directionally transferred to IRES/PDC316.The resulted plasmid IRES-IL-2/PDC316 was then treated with Salâ… ,and IRES-IL-2 gene product was separated and directionally cloned into CS+X^-/PDC316 to construct bi-expressive recombinant CS+X^--IRES-IL-2/PDC316.Part three Effects of Replication-Defective HBV-Recombinant on Wild-Typed Virus in vitroHBV genome without package signal from plasmid PCH3143 was cloned into PDC316 to construct adjuvant plasmid 3143/PDC316.Recombinant CS+X^--IRES-IL-2/PDC316 and adjuvant plasmid 3143/PDC316 were embedded with lipidosome and then were co-transfected into HepG2 cell line,with transfection of recombinant CS+X^--IRES-IL-2/PDC316,wild-typed 1.3-fold HBV/PDC316,plasmid 3143/PDC316 and adenovirus vector as three independent controls.Toxicity of recombinant or plasmid was determined by MTT assay after transfection for 48 hours. The replication and package ability of the HBV recombinant were assessed by:1) determination of intracellular expression of C-S fused protein by RT-PCR,and determination of IL-2 in the culture medium by double-antibodies-sandwich ELISA,2) extraction and detection of viral particle in the culture medium by PCR.Our result indicated that:1) no obvious toxicity of replication-defective HBV recombinant to HepG2 cells was observed,2) transfection of the recombinant could express C-S fused protein in the HepG2 cells and could express and excrete IL-2 to the supernatant, but could not package and excrete hepatic B viral particles to culture medium unless it was co-infected with adjuvant plasmid 3143/PDC 316.To observe the effect of replication-defective HBV-recombinant on wild-typed virus,HepG2 cells were separately co-transfected with:1) CS+X^-/PDC316 and wild-typed 1.3-fold HBV/PDC316,2) CS+X^--IRES-IL-2/PDC316 and wild-typed 1.3-fold HBV/PDC316,3) PDC316 and wild-typed 1.3-fold HBV/PDC316.Forty eight hours after co-transfection,intracellular HBV and HBV in the culture medium were determined by quantitative PCR.Results:Intracellular HBV was 6.45×10⁶copies/ml,5.75×10⁶copies/ml and 2.23×10⁷ copies/ml in the three groups,so the recombinant plasmid CS+X^-/PDC316 and CS+X^--IRES-IL-2/PDC316 co-transfected with 1.3-fold HBV/PDC316 could decreased intracellular synthesis of HBV DNA(71.08%and 75.29%).Respectively, HBV in the culture medium was 4.06×10⁶copies/ml,5.06×10⁶copies/ml and 1.87×10⁷ copies/ml,and HBV DNA in the culture medium had been lowered by 74.20%and 72.94%.Our result indicated that replication-defective HBV-recombinant could significantly inhibit the replication and excretion of wild-typed hepatitis B virus.Conclusions:In this research,a novel replication-defective HBV recombinant, which could express and excrete IL-2,was successfully based on adenovirus vector.It could replicate and package virons if it was co-transfected with another HBV plasmid which lacked package signal sequence.But if it was co-transfected with wild-typed hepatitis B virus,it could significantly the replication and excretion of the wild-typed hepatitis B virus.