Study Of The Antiviral Effect On Duck Hepatitis B Virus Of Recombinant Duck Interferon-α

In the article, expression conditions of engineering E.coli expressing recombinant duck interferon-a and antiviral effect on duck hepatitis B virus of recombineant duck interferon-a were studied,the results showed as follow:1.The expression conditions of engineering E.coli expressing recombinant duck interferon-α: The expression conditions of the genetic engineering E.coli BL21(DE3)/pET32a+-DuIFN-αwhich expressed duck interferon-a,including IPTG concentration, induction time, harvest period, cultivation temperatures, glucose concentration and culture medium were optimized. The optimal inductive conditions were as follows: the culture medium was TB+5g/L glucose, the culture temperature and the induction temperature were 37℃and 30℃,OD₆₀₀ was 0.8～1.1,the concentration of IPTG was 0.2mmol/L and the induction time was 4-5h.By optimizing way,the relative amount and total amount of duck interferon-a was 34.1% and 79.9 respectively.2.The antiviral effect on duck hepatitis B virus of recombinant duck interferon-α:The expression product was purified and refolded by metal immobilization affinity chromatography(MIAC) and the anti-VSV activity of recombinant duck interferon-αwas up to 1.28×10⁴U/mg.One-day-old DHBV-negative Penkin ducklings were inoculated with 10⁹ viral genome equivalent of DHBV by intravenous injection. On the 7th day after being infected with DHBV, DHBV-positive duckings administrated recombinant duck interferon-a with the dose of 500U,1000U,2000U per duck respectively by intramuscular injection(IM) every other day for 15 days.The trail was compared with lamivudine and normal saline. The number of DHBV DNA in serum was detected by fluorsescence quantitative polymerase chain reaction(FQ-PCR) before treatment,5 days, 10 days, 15 days after treatment and 3 days after withdrawal. Liver, pancreas,kidney, spleen, lung, cardiac muscle, brain and duodenum were collected at the same time.The content of DHBV DNA in these tissues were detected by FQ-PCR, the results indicated:Lamivudine showed a rapid inhibiting effect on DHBV DNA, but this effect last a short time, and the serum level of DHBV DNA increased again after withdrawal. The serum DHBV DNA levels all dropped after treatment in DuIFN-αtreated groups. The serum DHBV DNA levels in DuIFN-α2000U group on the 5th, 10th and 15th day of treatment were obviously lower than the level before treatment, there were significant differences(P≤0.05). The serum DHBV DNA levels in DuIFN-α1000U group on the 10th and 15th day of treatment were obviously lower than the level before treatment, there were significant differences(P<0.05). DuIFN-αat the dose of 500U also showed certain inhibiting effect on DHBV DNA,but there were no statistical significance as compared with serum DHBV DNA levels of before treatment. After withdrawal of DuIFN-α, the inhibiting effect in DuIFN-α1000U group continued but levels of serum DHBV DNA in 2000U and 500U groups rebounded.Recombinant duck interferon-αcut down markedly the serum level of DHBV DNA and can restrain the duplication of DHBV in vivo. Moreover the inhibiting effect correlates with dose of DuIFN-αand treatment time. Although this effect DuIFN-αis weaker than that of lamivudine, it continues longer in DuIFN-α1000U group.Recombinant duck interferon-a droped markedly levels of tissue DHBV DNA, but the inhibiting effect had difference in different tissues. After medicated with DuIFN-αat dose of 1000U 15 days, copies of DHBV DNA in pancreas, brain, heart, lung and duodenum were lower one order of magnitude above than these in normal saline group, and copies of DHBV DNA in liver,spleen and kidney lower 0.2-0.5 order of magnitude as compared with these in normal saline group.