Construction Of Toxoplasma Gondii ROP46gene Deletion Mutant And The Analysis Of Its Biological Characteristics

Rhoptry proteins of T.gondii are important secreting proteins, which are related to the invasion and replication of the parasites. Rhoptry proteins participate in the formation of parasitophorous vacuole membrane, regulate host cell gene expression and virulence. It was reported that the expression of Rop46 in the acute stage of infection was significantly different from that in chronic infection stage. Otherwise, the expression of ROP46 had significant difference in the different virulence of T.gondii invasion. ROP46 may be associated with the formation of parasitophorous vacuole membrane and T.gondii’s invasion to host cells.Therefore the research of ROP46 protein contribute to studying the mechanism of toxoplasma tachyzoite transforming to bradyzoite and developing drugs that prevent and theat toxoplasmosis.With the aid of PCR technology and prokaryotic expression technology ROP46 protein was successfully obtained. The reaction degree of T.gondii positive serum and ROP46 protein was detected by ELISA method. we generated the Rop46 gene deleted T.gondii(T.gondii /△Rop46)using CRISPR/Cas9 technology with the Rop46 gene knockout recombinant plasmid. Flow cytometry and counting method were used to analyze the growth characteristics of ROP46 gene deleted T.gondii. Animal experiment was used to detect the T.gondii /△Rop46 toxicity.ELISA test results showed that ROP46 and T.gondii serum are positive reaction. Sequencing analysis showed that a 82 bp sequence was deleted from the Rop46 gene of T.gondii /△Rop46strain, proving that Rop46 gene deleted T.gondii(T.gondii /△Rop46) was successfully generated.The analysis of the growth characteristics of T.gondii /△Rop46 by flow cytometry proved that invasion and replication of T.gondii /△Rop46 significantly decreased comparing to the parental T.gondii strain, which demonstrated that Rop46 gene had significant impact on the replication and invasion of the parasite. Animal experiments showed that the rop46 gene had a effect on the virulence of T.gondii.Rop46 gene deleted T.gondii(T.gondii / △ Rop46) which can be inherited stably was successfully generated by CRISPR/Cas9 technology. And what was confirmed is that ROP46 has a significant effect on the invasion replication and virulence of T.gondii.