Studies On NcSRS2 DNA Vaccine Of Neospora Caninum

Neospora caninum, an apicompexan prototoan parasite, causes neurological disease in dogs and is a major cause of infectious abortion in cattle. Currently, there is no effective method for control of neosporosis. Generally speaking, immunoprophylaxis to prevent abortion or congenital transmission is feasible goal. Therefore, we studied the efficacy of NcSRS2 DNA vaccine.Based on the NcSRS2 gene sequence of Neospora Caninum, a pair of primers containing Kozak sequence, Pst I and Xba I enzyme digestion sites were designed. Using the Plasmid which contains NcSRS2 gene,The ORF region of NcSRS2 gene was amplified by Polymerase chain reaction (PCR), then the PCR product was digested with Pst I and Xba I, and the eukaryotic expression vector pcDNA3.1(+)was also done ,then the gene was cloned into the vector. Finally a recombinant plasmid named pcNcSRS2, which was identified by PCR, restriction enzyme analysis and sequencing, was obtained.The recombinant plasmid pcNcSRS2 was extrcated with an endotoxin-free plasmid purification kit. Following transfection of pcNcSRS2 into mammalian cell Vero with liposome technique, the expression of NcSRS2 gene was examed with RT-PCR and western-blot. The results of RT-PCR and western-blot demonstrated that the recombinant plasmid pcNcSRS2 that we have constructed before expressed NcSRS2 antigen in mammalian cell Vero, and laid foundation for researches on DNA vaccine based on NcSRS2 gene.The BALB/c mice were immunized by intramuscularly injection with saline solution, non-coding plasmid DNA pcDNA3.1(+), pcNcSRS2, the complex of pcNcSRS2 and complete Freund's adjuvant(CFA) respectively. The results of ELSIA, SIs, NO level and semi-quantitative RT-PCR analysis of IL-2 and IFN-γmRNA indicated that constructed DNA vaccine expressing NcSRS2 can result in the preferential development of Thl type of immune responses and a weak humoral immune response in DNA vaccine-immunized mice; Moreover, to a certain extent, the use of complete Freund's adjuvant can improve these responses .