Eukaryotic Expression Of Neospora Caninum Jilin Strain SAG1 Gene And The Evaluation Of Immunology

Neosporiasis is a protozoal diseases which by Neospora caninum parasites in a variety of animal cells caused pregnant domestic animal miscarriage, stillbirth and neonatal nervous system disorders of fetal movement. The disease has caused enormous economic losses to animal husbandry which is especially serious harm to the cattle. As there is yet no effective therapeutic drugs and vaccines to control the occurrence and development of the disease, so vaccine research is the hot issue of Neosporiasis currently studying.Based on the SAG1 gene sequence of Neospora caninum in the Genbank (AF132217), a pair of primers containing Kozak sequence, start codon, stop codon, Bam H I and Eco R I enyme digestion sites were designed and synthetized. SAG1 gene was amplified by PCR, cloned into pMD18-T simple vector, identified by PCR and double-digestion and then analysesed by its sequence. The correct SAG1 gene was cloned into pVAX1 eukaryotic expression vector and then transfected into BHK21 cells after PCR, double-digestion and sequence analysis. The expression condition of the recombinant plasmid was detected by IFAT and RT-PCR. BALB/c mice were immunized by the recombinant plasmid pVAX-SAG1. The level of humoral immunity and cell mediated immunity was detected by indirect ELISA and flow cytomertry.The result indicated that SAG1 gene was 780bp and encodes 260 amino acids. The homology of nucleotide sequence between the cloned SAG 1 gene and American strain (AF132217) was 99%. The homology of amino acid sequence was 98.8%. It was proved by IFAT and RT-PCR that the recombinant plasmid was transiently expressed. The level of humoral immunity was significantly higher than pVAXl immunity class and PBS control group (P<0.01). The level of cell mediated immunity was significantly higher than PBS control group (P<0.05). This research developed a new way to prepare the nucleic acid vaccine against Neospora caninum.