Immuno-enhance Effect On IBDV Inactivated Vaccine By Chicken IL-18 Prokaryotic Expression Protein And Eukaryotic Expression Plasmid

Interleukin 18(IL-18) has multiple biological activities including induction of IFN-γfrom NK cells and antigen- or mitogen-stimulated Th1 cells, upregulation of IL-2R on T cells, enhancement of Fas ligand-mediated cytotoxicity of T-helper cells and augmentation of NK cell cytotoxicity. These on human beings and animals were identified by many documents recently. Chickens IL-18 was constructed to recombinant chicken IL-18 genes in our previous study in 2003. The bioactivities of expressed product of ChIL-18 in E.coli would be assayed and the methods of purification and renaturation will be assessed and the effect of it on the immune in chickens will be study as following.1 Artificial Chaperone-assisted Refolding of Recombined Chicken Interleukin-18 Gene In E.coliTo study the technique of boosting the renaturation yield of rChIL-18 by using aritificial molecular chaperone composed of cetyl trimethyl ammonium bromide(CTAB) andβ-cyclodextrin(β-CD).The recombinant plasmid of mChIL-18 prokaryotic expression was transformed into E.coli BL21(DE3) strain and then induced by IPTG at 37℃. The recombinant mChIL-18 was expressed efficiently in inclusion bodies in E.coli. After crushed and washed, the inclusion bodies were thoroughly denatured with 6 mol/L of guanidine hydrochloride.,and then the artificial molecular chaperone was used to promote protein refolding. After the rehabilitation of products was purified by bag filter, its activity was detected by lymphocyte proliferation assays. The SDS-PAGE analysis indicated the expressed ChIL-18 protein had molecular weight of 44000.The expressed product existed in the form of inclusion body.Two protein bands of Mr 44000 and 26000 appeared on SDS-PAGE gel.The percentage of renaturation was 42.54 with artificial molecular chaperone.The results of MTT assay showed the expression of ChIL-18 protein in E.coli BL21(DE3) greatly induced the proliferation of chicken T lymphocytes. The artificial chaperone technique can obviously boost the renaturation yield of rChIL-18. The purified and expressed product of fusion chicken Interleukin-18 gene in E.coli have relativity high bioactivity.2 The Study On Influencing Factors In The Refolding Process of artificial Molecular Chaperone Assisting Chicken IL-18 Recombination Protein The recombinant plasmid of mChIL-18 prokaryotic expression was transformed into E.coli BL21(DE3) strain and then induced by IPTG at 37℃..After crushed and washed the expressing inclusion bodies were thoroughly denatured with 6 mol/l guanidine hydrochloride.Then according to experiment design,the effects of rChIL-18 protein refolding yield on different densities are investigated by the systems of artificial chaperone on different densities.Experiment results indicate that there is a optimal condition on assiting rChIL-18 protein by using the artificial chaperone technique. The optimal condition can improve the refolding yield of rChIL-18 protein,and then the expressed product of fusion chicken IL-18 gene in E.coli have relativity high bioactivity.3 The Study of IBDV Inactivated Vaccine's Immunologic Enhancement Which Is Caused by Chicken IL-18 Prokaryotic Expression Protein and Eukaryotic Expression PlasmidTo study the immunologic enhancement of IBDV inactivated vaccine which is caused by Chicken IL-18 prokaryotic expression protein and eukaryotic expression plasmid pcDNA3.1TOPO-mChlL18,two tests about diction of neutralization antibody and ConA proliferative response of T lympholeukocyte are needed. There are distinguished difference(P<0.05) between the protein class,the plasmid class and the vaccine class on antibody level after post inoculating 21,28,35 and 42 days, moreover the protein class is more obviously than the plasmid class; The result of proliferative response of T lympholeukocyte is similar to antibody detect test. The class of simultaneous inoculation of chicken IL-18 prokaryotic expression protein and eukaryotic expression plasmid gains 93.3﹪conservation ratio while the vaccine class gains 73.3﹪.Results display: chicken IL-18 prokaryotic expression protein and eukaryotic expression plasmid have the obviously ability to enhance the Immunologic effect of IBDV inactivated vaccine.ConclusionThe artificial chaperone technique can obviously boost the renaturation yield of rChIL-18. The purified and expressed product of fusion chicken Interleukin-18 gene in E.coli have relativity high bioactivity.Experiment results indicate that there is a optimal condition on assiting rChIL-18 protein by using the artificial chaperone technique. The optimal condition can improve the refolding yield of rChIL-18 protein,and then the expressed product of fusion chicken IL-18 gene in E.coli have relativity high bioactivity.Results display: chicken IL-18 prokaryotic expression protein and eukaryotic expression plasmid have the obviously ability to enhance the Immunologic effect of IBDV inactivated vaccine.