Study On Molecular Diagnosis Methods And Gene Immunity For Swine Toxoplasmosis

Toxoplasmosis causes reproductive failure in pregnant women and pregnant animals, manifested as puny, and abnormal embryo, abortion and stillbirths. The Toxoplasma gondii is ubiquitous among people and animals throughout the world and is high infection in most herds. It is the enemy of human prepotency and one of the four factors leading to abnormal embryo in pregnant women. Because of above reasons, establishing quick diagnostic metheds and developing a DNA vaccine of high efficancy and security are very important to prevent and controll toxoplasmosis. In this research, MIC3 and ROP2 of Toxoplasma gondii were highly expressed in E.coli; rMIC3-ELISA and rMIC3-LAT diagnostic metheds for swine toxoplasmosis were first established to our knowledge; Then the MIC3 gene was cloned, sequenced, expressed in eukaryotic IBRS-2 cells and the possibility of DNA vaccine were studied too. This research will be useful for vaccination, diagnosis, prevention and control of swine toxoplasmosis.MIC3 and ROP2 of Toxoplasma gondii are consided as impotant candidates of diagnostic methods and DNA vaccine. The MIC3 related to Toxoplasma gondii infection are expressed during tachyzoite, bradyzoite and sporozoite. The study on MIC3 is little reported. The primers were derived from the DNA sequences that have been published in GenBank. The entered ORF of MIC3 and major ORF of ROP2 gene were amplified from the total DNA of Toxoplasma gondii RH strain and cloned into pGEX-KG vector respectively, the recombinant plasmid pGEX-KG-MIC3 and pGEX-KG-ROP2 were constructed. The gene sequences of amplified fragments were determined and compared in GenBank by DNA Blast and Protein Blast. The nucleotide sequences of MIC3 and ROP2 were found to be similar to the MIC3 and ROP2 from GenBank and the homology of their amino acid sequences were high over 99 % respectively. Then both plasmids were respectively introduced into E.coli-BL₂₁-CodonPlus. After induction by IPTG, high expressions were all found. The expressed products of MIC3 and ROP2 could specifically bind to the antibody against Toxoplasma gondii by Western-blot.The quick diagnostic methods of ELISA and LAT for swine toxoplasmosis were established by rMIC3. After detected tests of positive sera of other pathogen, serum dilutions of Toxoplasma gondii and batches (the same/different), store times of rMIC3-ELISA and rMIC3-LAT products in 4℃, the results indicated that both methods had high specificity, good sensitivity, stability and longer shelf life of products in 4℃.rMIC3-ELISA and rMIC3-LAT could detect the dynamics of the antibody levels after the swines were primary inoculated hypodermically Toxoplasma gondii RH strain. The results revealed that the antibody against Toxoplasma gondii was first detected by LAT till 4-6 day and till 14-16 day by ELISA; the peak of the antibody was detected by LAT till 14-31 day and till 24-31 day by ELISA; then the antibody begun to decrease and could be detected by LAT till 64 day and could not be detected by ELISA, after the swines primary challenged with Toxoplasma gondi RH strain. Besides, rMIC3-ELISA and rMIC3-LAT had detected 471 samples from clinical swine sera, the accordance of both methods was 92.56 %.The entered ORF of MIC3 and IFN-γgene were cloned into the the eukaryotic expression vector of pcDNA3.1(+), resulting recombinant expression plasmids of pcDNA-MIC3 and pcDNA-IFN-γ. Then these plasmids divided into five groups were transfected into IBRS-2 cells. Expressed proteins of pcDNA-MIC3 and pcDNA-MIC3+ pcDNA-IFN-γwere detected by ELISA and located in cell. On the basis of above work, these plasmids divided into five groups were used as DNA vaccines to immune mice. The results showed that the pcDNA-MIC3 and pcDNA-MIC3+pcDNA-IFN-γplasmids could induce specific cell-mediated and humoral immunity. The immune responses induced by co-immunization with pcDNA-MIC3 and pcDNA-IFN-γwere stonger than that induced by pcDNA-MIC3. This sugested plasmid pcDNA-IFN-γcould enhance induced immunity effect to pcDNA-MIC3.After observoving of clinical symptoms and survivable days of Balb/c mice immunity protect against T.gondii RH strain, the results revealled that the Balb/c mice induced by pcDNA-IFN-γcould slightly resist to Toxoplasma gondii RH strain, the Balb/c mice induced by pcDNA-MIC3, pcDNA-MIC3+pcDNA-IFN-γcould strongly resist to Toxoplasma gondii RH strain.In this research, the diagnostic methods of rMIC3-ELISA and rMIC3-LAT of swine toxoplasmosis have been established, and the eukaryotic expression and gene immunity of MIC3 have been studied, these results indicate that MIC3 has a bright prospect as study of clinical diagnostic methods of toxoplasmosis and further developing DNA vaccine.