The Expression Of Neospora Caninum NcGRA2 And The Evaluation Of Its Effects On ELISA Diagnostic And Immunoprotection

Neosporosis is a kind of protozoa desease that is produced by Neospora caninum being parasitic in animal cells.The disease is widely popular in the world and the main clinical symptoms include fetation miscarriage,stillbirth and weak fetus,which bring some economiclosses to the poultry industry.At present the effective drugs and vaccines controlling neosporosis does not yet exist.The early diagnosis and prompt isolation and elimination of the infected cattle are the main method avoiding neosporosis prevalence.In addition,the vaccine development of neosporosis is an urgent matter.The ELISA early diagnosis method for neosporosis basing on the study of N.caninum NcGRA2 will be established and the recombinant protein subunit vaccine and nucleic acid vaccine of neosporosis will be preparated in this study.Also,the combined immune protection effect of recombinant protein subunit vaccine and nucleic acid vaccine will be tested in the study.The sudy will provide a scientific basis for the prevention of neosporosis occurrence.A pair of primers containing EcoRI and XhoI restriction enzyme site was designed according to the NcGRA2 gene sequence of N.caninum.The total RNA of N.caninum Nc-1 strain was extracted and the complete ORF gene fragment of NcGRA2 was amplified by RT-PCR.The gene fragment of NcGRA2 was joined to the T-vector pMD20 to genernate the recombinant plasmid pMD20T-NcGRA2 by T-A cloning.The positive clones were identified by enzyme digestion.The sequencing and biological analysis showed that the NcGRA2 gene sequence of Nc-1 strain was identical with that of Ncliverpool strains and had the 49%homology of amino acid sequence of Toxoplasma gondii TgGRA2.The prediction of encoding amino acid of NcGRA2 gene indicated that the NcGRA2 protein had good antigenicity,stability and hydrophilicity,which would benefit the purification of protein.A pair of primers were designed for the amplification of NcGRA2t being the part of removed the N terminal signal peptide fragment of N.caninum NcGRA2 gene.The pMD20T-NcGRA2 was used as the template for the PCR amplification.The NcGRA2t fragment was connected into the prokaryotic expression vector pGEX-4T 1 and constructed the prokaryotic expression vector.The induced expression of E.coli and SDS-PAGE analysis showed that the NcGRA2t protein existed in the supernatant and inclusion body of the culture.The Western blot analysis indicated that the NcGRA2t protein could be recognized by the serum of dog infected by N.caninum.The polyclonal antibodies against NcGRA2t were prepared by injecting NcGRA2t protein into the BALB/c mice.The NcGRA2t polyclonal antibody could react with the lysate antigen of Neospora,but not with the lysate antigen of Toxoplasma gondii.The indirect ELISA method for diagnosis of neosporosis was established using the NcGRA2t as the diagnostic antigen.The NcGRA2t diagnostic antigen could detect mouse serum infected by neosporosis,but did not react with mouse serum infected by T.gondii.This showed that the ELISA method could distinguish between Neospora infection and the infection of T.gondii.The NcGRA2t diagnostic antigen were compared with the NcS AG1t diagnostic antigen by detecting the N.caninum serum,and the result revealed that the indirect ELISA method of NcGRA2t diagnostic antigen could be used for the early diagnosis of neosporosis.The ELISA result of detection the field samples proved that the ranch dogs had higher chances than the pet dog to infect neosporosis.The NcGRA2 fragments from pMD20T-NcGRA2 vector were cut using EcoRI and XhoI and attached to the eukaryotic expression vector pcDNA3.1 to construct the recombinant plasmid pcDNA3.1/NcGRA2.The PcDNA3.1/NcGRA2 recombinant plasmid was transfected into 293 cell by liposome and the expression of NcGRA2 protein was detected by indirect fluorescent antibody test.The result indicated that the pcDNA3.1/NcGRA2 could express foreign protein in mammalian cells and also laid the foundation for the research of nucleic acid vaccine of Neospora NcGRA2.The BALB/c mice were immunized with NcGRA2t recombinant protein vaccine and nucleic acid vaccine of pcDNA3.1/NcGRA2.The IgG,IgG1 and IgG2a humoral immune response,IFN-y and IL-4 cell immune response and Thl/Th2 type immune response could be induced in mice and could reduce the N.caninum infection to the mice brain.