Study On The Bioactivities Of Recombinant Chicken Interleukin-6Protein (rCHIL-6) With Different Concentrations In Vitro And Vivo

Studying on the biological activity of recombinant protein of rChIL-6with different concentration in vitro and vivo，which would predict the best concentration of immune function of rChIL-6in vivo by detecting its activity in vitro，lay the theoretical foundation of rChIL-6for industrial production and agent to guide clinical.The following tests were performed in the present study in vitro：Sandwich Elisa method was used to detect if the rChIL-6has the specific immune activity with rChIL-6monoclonal antibodies and MTS method was employed to determine the immune enhancement of different concentrations rChIL-6on proliferation activities of chicken lymphocytes in spleen and the peripheral blood,selecting4rChIL-6protein concentration(stoste、1:23dilution、1:25dilution、1:27dilution) in vivo experiment.In the study in vitro: the immune-enhancing test of rChIL-6protein on IBDV were carried out by inoculating rChIL-6protein and IBD inactivated vaccine and live vaccine in16037-day-old SPF chicken divided into twenty groups averagely. The serum antibody against IBDV, the expression levels of chicken IL-2、IL-4、IL-6、IFN-α and IFN-γ,CD4+/CD8+value the proliferation activities of lymphocytes in spleen and the peripheral blood were detected to select the best concentration of rChIL-6which had better immune function and the maximum dilution in vivo. According to the above results, the immune-enhancing test of rChIL-6protein on ND were carried out by subcutaneous injection and intranasal instillation method inoculating rChIL-6protein and ND inactivated vaccine and live vaccine in48130-day-old SPF chicken divided into six groups averagely to verify the best concentration of immune function of rChIL-6，to reveal its effection on the serum antibody against NDV、lymphocyte subsets and immune enhancement of different inoculation methods on NDV attenuated live vaccine. Result: In vitro, the rChIL-6has the specific immune activity with rChIL-6Mab，the rChIL-6protein content in rChIL-6was about121.57pg.mL-1，and it could distinctly improve the profileration activities of both spleenic lymphocytes and peripheral ones. rChIL-6protein solution dilution degree≤212, rChIL-6protein had significantly proliferation activity of chicken spleen lymphocytes and its OD490value significantly higher than those in Cell control group(p<0.01),≤25, different concentrations of rChIL-6protein has a similar stimulating spleen lymphocyte activity(p>0.05)；rChIL-6protein solution dilution degree≤1：211，rChIL-6protein had significantly proliferation activity of chicken peripheral lymphocyte, rChIL-6protein solution dilution degree=27, its OD490value had the highest value. Relatively speaking, the proliferation activity of chicken splenic lymphocytes was significantly higher than that in peripheral blood T lymphocytes.In vitro, the expression levels of chicken IL-2, IL-4, IL-6, IFN-α,IFN-γ, CD4+/CD8+value and serum antibody against IBDV，proliferation activity of chicken spleen lymphocytes and peripheral blood (7-35days post-immunization，DPI) of different concentrations rChIL-6protein（stoste、1:23dilution、1:25dilution） and IBD inactivated vaccine coimmunized groups、 rChIL-6protein and IBD live vaccine co-immunized groups were significantly higher than that of their corresponding IBD inactivated vaccine control groups and their corresponding IBDV live vaccine control groups (P<0.05);The expression levels of chicken IL-2, IL-4, IL-6,CD4+/CD8+value， proliferation activity of chicken spleen lymphocytes and peripheral blood (7-35days post-immunization，DPI) of rChIL-6protein control groups (2~5group) were significantly higher than that of PBS control group(1group)(P<0.05),but its expression levels of chicken IFN-α and IFN-α did not significant change. rChIL-6protein solution dilution degree≤1：25,these testing indexs above was similar to rChIL-6protein stoste group,s,but the rChIL-6protein1:27dilution control group,s significantly decreased; Relatively speaking, these testing indexs above of different concentrations rChIL-6protein and IBD inactivated vaccine coimmunized groups were slightly better than rChIL-6protein and IBD live vaccine co-immunized groups, and rChIL-6protein and IBDV attenuated live vaccine used separately groups, were slightly better than RChIL-6protein attenuated live vaccine pre mixed use group,.The rChIL-6protein1:25dilution and ND inactivated vaccine coimmunized groups、rChIL-6protein and ND live vaccine co-immunized groups could improve HI1~3antibody titers and proliferation activity of chicken spleen lymphocytes and peripheral blood significantly; Relatively speaking, these testing indexs above of different concentrations rChIL-6protein and IBD inactivated vaccine coimmunized groups were slightly better than rChIL-6protein and IBD live vaccine co-immunized groups, and rChIL-6protein and IBDV attenuated live vaccine used separately groups, were slightly better than rChIL-6protein attenuated live vaccine pre mixed use group,.Conclusion：the rChIL-6has the specific immune activity with rChIL-6Mab，significantly effection on proliferation activities of chicken lymphocytes in spleen and higher then that of chicken peripheral blood；In vitro, different concentrations rChIL-6protein and IBD/ND inactivated vaccine coimmunized、rChIL-6protein and IBD/ND live vaccine co-immunized could enhance both the expression level of the Th1, Th2type cytokines、CD4+/CD8+ratio and proliferation activity of chicken spleen lymphocytes and peripheral blood，to exert the cellular and humoral immune enhancement.;The enhancing effect of rChIL-6protein on humoral immunity was relatively higher than the cell immunity; the effect of inactivated vaccine was relatively higher than live attenuated vaccine; rChIL-6protein and IBD/ND attenuated live vaccine used separately were slightly better than pre mixed use. A correlation between rChIL-6protein in vitro and in vitro activity, rChIL-6protein1:25dilution had the maximum dilution which also had a smilar immune enhancement to stoste; the detection results of rChIL-6protein activity in vitro could be a basis for rChIL-6protein concentration selection in vitro...