Construction of fusion gene harbouring Ea3-1E and mChIL-15 and immune efficacy of its eukaryotic expression plasmid in SPF chickens.

The fusion gene,Ea3-1E-linker-mChIL-15,was constructed by co mbining 3-1E gene fragment encoding sporozoites surface antigen of Eime ria acervulina(Ea3-1E)and the gene fragment encoding m ature chicken interleukin 15(mChIL-15)with four flexible linker peptides SPGS by gene splicing overlap extension PCR technology.The eukaryotic expression plas mid pcDNA3.1-Ea3-1E-linker-mChIL-15 was constructed success fully. The recombinant plasmid was then transfected into 293T cells with Ca3(PO 4)2,and the transient expression of objective protein was detected by indirect immunofluorescence and immunohistochemistry.The recombinant plasmid pcDNA3.1-Ea3-1E-linker-mChIL-15,pcD NA3.1-Ea3-1E and pcDNA3.1 were used to immunize SPF chickens o f 14-or 21-day old by injection intra-muscularly in thigh muscle,respectivel y.Except for the control group,each group as challenged with 5×104 E.acervulina sporulated oocysts on 28 days of age,and the immune efficac y was observed.The results showed that the fusion gene Ea3-1E-mChIL-15 was successfully constructed,and the transient expression product of this fusion gene could be detected after transfection in 293T cell a t hour 30 post-transfection.The plasmid pcDNA3.1-Ea3-1E-linker-mChIL-15 could significantly provide immune protective efficacy against Eimeria challenge after two immunizations,compared to p lasmid pcDNA3.1-Ea3-1E,such as improving relative weight gain rate(96.48%),reducing oocyst decrease ratio(68.35%),decreasing average lesion sc ore in duodenum and so on.The anti-coccidial index was 183.78.