The Protective Efficacy Of Rhoptry Neck Protein 5 And 10 Genes Of Toxoplasma Gondii And Construction Of Their Gene Knockout Strains

Toxoplasma gondii is an intracellular protozoan parasite, infecting almost all vertebrates, causing toxoplamosis. Nearly one third of the human population has been infected by T. gondii in the world, and causes serious health problem to humans and animals. Currently, there are no effective medicines for toxoplasmosis in humans and animals, so immunoprophylaxis is the best way to prevention and control of toxoplamosis. But nowadays, there are no effective vaccines against toxoplasmosis for humans yet.The rhoptry is a subcellular organelle of apicomplexan parasite, it secretes rhoptry neck proteins(RONs) and rhoptry proteins(ROPs), which are different from their locations in rhoptry. It was estimated that there are 8 RONs in T. gondii, including RON1, RON2, RON3, RON4, RON5, RON8, RON9 and RON10, respectively. Most of RONs plays an important role in the invasion of T. gondii.The first part of our study examined sequence variation in the rhoptry neck protein 10(RON10) gene among 12 T. gondii isolates from different hosts and geographical locations。 After using PCR amplification, sequence analysis, and phylogenetic reconstruction,our results revealed that a low sequence variation in RON10 gene among the 12 T. gondii isolates, and it is not a suitable genetic marker for the differentiation of T. gondii isolates from different hosts and geographical locations, but may represent a potential vaccine candidate against toxoplasmosis.The second part of our study generated two eucaryotic plasmids of p VAX-RON5 p and p VAX-RON10 and used to immune BALB/c mice. We evaluated humoral and cellular immune responses and the protective efficacy against RH and PRU infections of T. gondii after 3 times vaccination. Both humoral and cellular immune responses were evoked in mice by two vaccine immunization, and p VAX-RON5 p DNA vaccine can induce partial protective immunity against acute and chronic T. gondii infections, but p VAX-RON10 failed. RON5 gene may represents an antigen candidate in developing practical vaccines of T. gondii.The third part of our study constructed two gene knockout plasmids Cas9-sg RON5 and Cas9-sg RON10 of RON5 and RON10 genes in T. gondii. Isolating maxiprep plasmids after sequencing verification, mixed with RH tachyzoits of T. gondii in appropriate proportion, transfected by electroporation in 1700 V,130 μs, 2 times of electric pulse, transfer transfections to Vero cells. Stable transfectants were selected for pyrimethamine after 72 hours growth of transfected parasites, then transferred parasites to 96-well plates by limiting dilution, PCR amplified and sequencing small segment of target sequence after monoclonal parasite was amplified and cultured. Our results indicate that it is difficult to gain the △RON5 directly, and △RON10 parasites exhibit no significant phenotype differences between with RH parasites.