Study On The Bioactivity Of Recombinant Chicken Interferon Alpha/Chicken Interleukin-2Fusion Protein In Vitro And The Optimal Dose Selection In Vivo

To clarify the biological activity of recombinant complex protein of rChIFN-α-Linker-ChIL-2in vitro and the optimal dose of inoculation in vivo to provide scientificbasis of rChIFN-α-Linker-ChIL-2as antiviral agents and immune enhancer in clinicalapplication，the following tests were performed in the present study：Sandwich Elisamethod was used to detect if the rChIFN-α-Linker-ChIL-2has the specific immuneactivity with rChIFN-α monoclonal antibodies（Mab） and rChIL-2Mab；50%cytopathic effect inhibition (CPEI) of CEF cells was used to examine efficacy ofrChIFN-α-Linker-ChIL-2and rChIFN-α control against vesicular stomatitis virus(VSV) and virulent strain LX of infectious bursal disease virus (IBDV)；MTS methodwas employed to determine the immune enhancement of the rChIFN-α-Linker-ChIL-2and rChIFN-α control on both spleenic and peripheral lymphocytes.37-day-old SPFchicken was artificial infected by virulent strain LX of IBDV，and selected rChIFN-α-Linker-ChIL-2and rChIFN-α protein which Anti-VSV activity units in CEF cells is200IU、1000IU、3000IU、5000IU by injection way to compare the activity in vivoto selected the optimal dose of inoculation of rChIFN-α-Linker-ChIL-2and rChIFN-αprotein in vivo；Through clinical observation、detected chicken′s detoxification rateat different time before and after the attack poison、IBDV dynamic distributed indifferent tissues and organs、the time of peripheral blood continued with IBDV、thecytokines（ChIL-2、ChIL-4、ChIL-6、ChIFN-α、ChIFN-γ）expression levels inperipheral blood、 spleenic and peripheral lymphocytes proliferative activity inchicken and differences in lymphocyte subsets and other indicators in peripheral bloodto reveal the mechanism of antiviral and immune-enhancing effects of rChIFN-α-Linker-ChIL-2and rChIFN-α protein in vivo，selected optimal dose of inoculation ofrChIFN-α-Linker-ChIL-2and rChIFN-α protein to play the best antiviral activity invivo. Flow cytometry was used to detected T lymphocyte subsets in peripheral blood at different time after the SPF chicken was injected in rChIFN-α-Linker-ChIL-2andrChIFN-α protein.Result： The rChIFN-α-Linker-ChIL-2has the specific immune activity withrChIFN-α Mab and rChIL-2Mab，the rChIFN-α protein and rChIL-2protein contentin rChIFN-α-Linker-ChIL-2was4.58×103pg.mL-1and1.06×103pg.mL-1；The anti-virus activities of rChIFN-α-Linker-ChIL-2against VSV and IBDV in CEF cells were8.707×105IU/mL and5.7926×104IU/mL，The anti-virus activities of rChIFN-protein control against VSV and IBDV in CEF cells were2.1469×105IU/mL and1.4482×104IU/mL，The of two kinds of protein against VSV was higher than thatagainst IBDV and rChIFN-α-Linker-ChIL-2was higher than rChIFN-proteincontrol； The rChIFN-α-Linker-ChIL-2was capable of distinctly improving theprofileration activities of both spleenic lymphocytes and peripheral ones， andrChIFN-α-Linker-ChIL-2was very significantly higher than rChIFN-protein control（p<0.01）. Different doses of rChIFN-α-Linker-ChIL-2had difference in anti-virusactivities in chicken. The duration of typical symptoms、case fatality rate、secretingvirus rate、tissue and organ infection rate and peripheral blood loading virus rate ofchickens injected with3000IU rChIFN-α-Linker-ChIL-2at different days postchallenge were28h、20％、22.2%、16.7%、23.5%，anti-IBDV antibody in serumlevels and other indicators were significantly lower than other doses of recombinantfusion proteins and rChIFN-α protein in control，expression of ChIL-2、ChIL-4、ChIL-6、ChIFN-α、ChIFN-γ were significantly higher than other groups；Spleenicand peripheral lymphocytes proliferation activity of different experimental groupsreached a peak at12dpc，the OD490value（0.2654、0.3406）of3000IU rChIFN-α-Linker-ChIL-2was significant higher than other experimental groups（p<0.05）；During12dpc to35dpc，the CD4+/CD8+of different doses of the experimental groupattack IBDV were significant lower than rChIFN-α-Linker-ChIL-2and rChIFN-αprotein control with the same dose，the CD4+/CD8+value of3000IU rChIFN-α-Linker-ChIL-2experimental group attack drug was significant higher than other doseand all the rChIFN-α protein control group attack drug；The above testing indicatorsshow that the antiviral activity of rChIFN-α-Linker-ChIL-2in vivo and vitro werehigher than the rChIFN-α protein control，and3000IU rChIFN-α-Linker-ChIL-2hasoptimal antiviral activity in chicken. After rChIFN-α-Linker-ChIL-2and rChIFN-αprotein apply for SPF chickens body（17d），the CD3+CD4+value rised、theCD3+CD8+value reduced and CD4+/CD8+rised sharply in peripheral blood；the CD4+/CD8+value of rChIFN-α-Linker-ChIL-2experimental group was significanthigher than rChIFN-α.Conclusion：The rChIFN-α-Linker-ChIL-2has the specific immune activity withrChIFN-α Mab and rChIL-2Mab；The rChIFN-α-Linker-ChIL-2protein has distinctanti-virus activities against IBDV and VSV，and enhances the profileration activitiesof both spleenic lymphocytes and peripheral ones in vitro tests and the profilerationactivities was significant higher than rChIFN-α control. Different dose of rChIFN-α-Linker-ChIL-2can play a non-specific anti-viral activity and immune-enhancingactivity through enhanced T lymphocytes and spleen lymphocyte proliferation inperipheral blood、improve the Th1，Th2cytokines expression levels and improve theCD4+/CD8+value；3000IU rChIFN-α-Linker-ChIL-2has optimal biological activityin chicken，the biological activity of different dose of rChIFN-α-Linker-ChIL-2inchicken was significant better than rChIFN-α protein control；The CD3+CD4+valuerised、the CD3+CD8+value reduced and CD4+/CD8+rised sharply in peripheral bloodafter rChIFN-α-Linker-ChIL-2apply for SPF chickens body，enhancing the cellularimmunity ability and the enhancing effect higher than the same dose of rChIFN-αprotein.