Examination Of Activities And Biologic Effects Of Recombinant ChIFN-γ, ChIL-18 And ChIL-2

The utilization of cytokines as therapeutic agents in livestock animals has become more feasible with the recent cloning of cytokine genes and the progression of new technologies.In this study, In order to examine the activities of recombinant ChIFN-γ,ChIL-18 and ChIL-2 expressed in E.coli. The cDNA of ChIFN-γ,ChIL-18 and ChIL-2 gene were subcloned into pMelBacB under the control of baculovirus polyhedrin to construct recombinant transfer vector pMelBacB-ChIFN-γ,pMelBacB-ChIL-18 and pMelBacB-ChIL-2 respectively. To obtain recombinant viruses that would express ChIFN-γ,ChIL-18 and ChIL-2, the recombinant transfer vector pMelBacB-ChIFN-γ, pMelBacB-ChIL-18 and pMelBacB-ChIL-2 were transfected into sf9 insect cells with wild-type Bac-N-Blue linear DNA using cationic liposomes respectively. The cultures were harvested, plaque-assayed with x-gal, and blue plaques were selected as recombinant viruses. After being purified for three times and identified by PCR, the purified viruses were named rBaculovirus-ChIFN-γ,rBaculovirus-ChIL-18 rBaculovirus-ChIL-2.Sf9 cells were infected with rBaculovirus-ChIFN-γ, rBaculovirus-ChIL-18 and rBaculovirus -ChIL-2 respectively and incubated at 28℃,the cultures were harvested at 24,48,72,96,120 and 144 hours post-inoculation respectively. The lysates of the cells were analyzed by SDS-PAGE, bands with a apparent molecular weight of 19kDa,23 kDa and 16 kDa for ChIFN-γ, ChIL-18 and ChIL-2 were observed respectively. To further confirm these bands, the lysates of sf9 insect cells infected with rBaculovirus-ChIFN-γ, rBaculovirus-ChIL-18 and rBaculovirus-ChIL-2 were analyzed by Western blot using polyclonal antibodies against ChIFN-γ, ChIL-18 and ChIL-2 expressed in E.coli, the results showed that these bands represented ChIFN-γ,ChIL-18 and ChIL-2 respectively.It was also demonstrated that the ChIFN-γ,ChIL-18 and ChIL-2 protein expressed in baculovirus expression system can show activities as well as their prokaryotic expression products. In order to examine in vivo biologic effects of ChIFN-γ,ChIL-18 and ChIL-2,chickens were treated with recombinant ChIFN-γ, ChIL-18 and ChIL-2 expressed in E.coli .The result showed that body weight gain enhanced in chickens treated with recombinant ChIFN-γ, ChIL-18 and up to 6.1% in weight gain trials.Furthermore, chickens treated with recombinant ChIFN-γ, ChIL-18 showed significant improvement in body weigh gain following IBV M41 challenge infection. Antibody titre in serum increased up to 1049.9 , IBV in trachea dispeared quickly and pathological changes were light in chickens treated with recombinant ChIFN-γ, ChIL-18 following IBV M41 challenge infection,but there is no significant change in chickens treated with recombinant ChIL-2.