| Toxoplasma gondii is an obligate intracellular protozoan that belongs to the phylum Apicomplexa parasites,which has a complex lifecycle. This parasite can infect all kinds of warm-blood animals including human, and cause serious zoonotic toxoplasmosis, it is known as the most successful parasitic protozoa. In this study we construct two mutants.(1) Construction of Stage-Specific Dual Fluorescent Transgenic Toxoplasma gondiiT.gondii tachyzoite invade into the host caused acute infection, and then the immune system or drugs worked, tachyzoite transform into bradyzoite and survive in the form of the tissue cysts, cause chronic infection which not exhibit any clinical symptoms.But when the immune system inhibition, such as AIDS patients or organ transplant patients, the bradyzoite transform back to tachyzoite cause clinical symptoms even death.This opportunity pathogenicity of T.gondii occurs due to the mutual conversion between tachyzoite and bradyzoite, but the transform mechanism is not clear yet.In general the methods of observe bradyzoite are fixed and stained, but they are effectiveness.As the transgene technology and fluorescent protein technology applied and developed in T.gondii, shows that the conversion between tachyzoite and bradyzoite with fluorescent is more easier to be observed in vitro.In this study we choose three genes: a tachyzoite stage specific gene SAG1, a bradyzoite stage specific gene BAG1, and the both stage has the gene GRA2, we searched and analyzed them go through the NCBI and ToxoDB(http://ToxoDB.org),and then determined the promoter of SAG1 and BAG1,the terminator of GRA2. We designed primers respectively, got the three sequences by PCR. Through using the overlap PCR, digest ligation, fused SAG1p-GFP-GRA2 t, and BAG1p-RFP-GRA2 t, and then put them into a plasmid pcDNA3.1(+) with a drug screening gene DHFR. Then used electro transformation to make the plasmid into T.gondii RH. After drug screening cultivate we got the stable green fluorescent mutant of T.gondii, but not got the red fluorescent. In our study we got the fused plasmid successfully, and use this plasmid we can get the mutant which can be observed more direct and easy in vitro, and it can provide convenient tools to study the interconversion of tachyzoite and bradyzoite and also can provide the research platform of new anti-T.gondii drugs.(2) Construction of knockout MIC3 Transgenic Toxoplasma GondiiThe reason why T.gondii can infect so many hosts is becouse of its successful invasive mechanism.In the process of the invasion the attachment is the key part. At this process the microneme protein(MIC) interaction with surface protein or sugar of host cell, mediate the parasite directional attach to the host cell.It shows the MIC is important to the pathogenicity of T.gondii.In this study we chose the MIC3 which is one of the MIC family to knock out.This gene codes a protein activin dimer which mediate the parasite attach to the host cell.We choose CRISPR/Cas9 system to knockout MIC3. We construct two plasmid: MIC3 knockout plasmid and DHFR homologous integration plasmid. And then according to upstream, middle, downstream sequence of MIC3 in ToxoDB, we design three pair primer to check the correct of the knockout. After the drug screening cultivate, we got the MIC3 deficiency mutant.Use This mutant we do the growth assay, plaque assay, virulence assay. To research the influence; after MIC3 deficiency to the T.gondii. Provide some experimental basis to further research on MIC3. Meanwhile maturate the CRISPR/Cas9 gene knockout technology in our lab to help study other gene function in T.gondii;... |
