Construction And Biological Characteristics Of TgCPB-deficient In Toxoplasma Gondii

Toxoplasma gondii is an obligated intracellular parasite, which can infect in more than200animal species, including humans, and one third of the world human population is chronically infected. Toxoplasmosis is an important zoonotic parasitic disease,which was caused by T. gondii. T. gondii infection in pregnant women can lead to spontaneous abortion, still-birth. For AIDS patients, the death caused by toxoplasmosis is ranked high on the list of disease. Toxoplasmosis has also causing economic losses in livestock. Parasite cathepsin can take participate in take nutrients from the host cells, infected host cells and take parasites evade the immune response from host cells. Toxoplasma gondii Cathepsin is a potential candidate factor diagnostic drugs and vaccines have been studied extensively. In view of, Toxoplasma gondii cathepsin B (TgCPB) played an important role in the invasion and proliferation of host cells, in this research, we studied part of the biological characteristics of TgCPB using the gene koockout technique, so as to provide the basis for preventing the Toxoplasmosis.so, studying the biological characteristics of TgCPB has a significance in prevention and control of toxoplasmosis.In this study, Viaing PCR to specifically amplified the5’untranslated region (1709bp) and3’ untranslated region (1810bp), and then cloned them into the pBluescript plasmid, respectively, at same time cloned into the against-phle gene, and structure the5’UTR-T-pBluescript-3’UTR-phle transfection plasmid. Restriction analysis results demonstrated that5’UTR-T-pBluescript-3’UTR-phle plasmids was successfully constructed, via the DNA sequence analysis, it is identical accordance with the homology of the genome of T. gondii which published in Genebank.Screening and identification of TgCPB-deficient Toxoplasma gondii:Digested the linearized recombinant plasmid using NotI enzyme, obtained a8088bp band,transferred the plasmid into Toxoplasma with the technique of electroporation, and recombined into the genome of Toxoplasma. Screened the positive clones on PHLE resistance medium, and the negative could not live on which, and received the TgCPB deleted Toxoplasma after1month,obtained the TgCPBdeficient strains.Identified by PCR, deletion strains successfully amplified2134bp,2552bp target band around the arm. According the Knockout (3238bp) genomic sequences, designed primers for Southern hybridization probes, after Southern-blotting identified, TgCPB deletion strains without band, GT1wild strains had bands, these two experiments mean the accomplishment of the constructing of TgCPB-deficient Toxoplasma gondii.The biological research on TgCPB-deficient Toxoplasma gondii:Researched on the adhesion, invasion of Toxoplasma TgCPB mutant by indirect immunofluorescence assay,Adhesion test showed that the TgCPB-deficient Toxoplasma did little influence on the adhesion capacity of host cell, indicating that the gene might not be directly involved in the process of adhesion host cell. Invasion experiments showed the same results.In this study, we also detected the proliferation of Toxoplasma gondii in host cell after deletion TgCPB gene by indirect immunofluorescence assay. The results showed that the proliferation of Toxoplasma gondii in host cell was significantly weakened after deletion TgCPB gene. In addition, the use of quantitative PCR method to detect the proliferation of the parasites, the results showed that the gene deletion TgCPB, Toxoplasma gondii proliferation was slowed significantly in cell, this accordance with the immunofluorescence experiments, and it is suggesting that this gene may be a new virulence gene in Toxoplasma, and may be involved in the pathogenesis of Toxoplasma gondii.We dye of TgCPB deletion strains and wild strains GT1by PI staining, and using flow cytometry techniques to detection the cell cycle. The results showed that the percentage of S phase GT1group was35.13±1.84, CPB group was53.03±1.87,which showed that TgCPB deletion mutant group advance into S phase, which has a longer duration time, it indicating that after the gene deletion of TgCPB, TgCPB has earlier entered a period of growth platforms than GT1, reducing the accumulation of parasite growth within a certain time, the result also demonrtrated that it is not conducive to the proliferation of Toxoplasma gondii after deletion TgCPB, accordance with the results of quantitative PCR results.TgCPB was speculated as a virulence gene by the results of quantitive PCR and the Flow cytometry assay.Toxoplasma gondii co-cultured with macrophages, detected the cell culture supernatant NO levels by Griess method, the results showed that the NO expression levels of TgCPB-deficient with significantly increased,was significant difference with the wild-type strain group by the biological software for analysis. This suggests that the TgCPB gene may play a very crucial role in T. gondii-mediated signaling pathways,the result provided the basis for further study of the function of the gene.In addition.we identified the location of CPB in Toxoplasma gondii, according to the published sequence in ToxoDB, designed and synthesized a pair of primers,expressed of recombinant proteins PET-22b-CPB-DE3by prokaryotic expression experiments. The recombinant protein was identified by SDS-PAGE and Western-blotting, confirmed to be about62.5kD in size,indicated the success of construct the recombinant protein. purified the protein by nickel column, immunized mice, polyclonal sera, immunofluorescence experiments were performed to locate the TgCPB with Toxoplasma.The result showed that the protein localization in both ends or surfaces of Toxoplasma. In this stuy, we constructed TgCPB-deficient successfully,by studying part of its biological characteristics we found that gene deletion of TgCPB had a significant impact in their normal life activities, which provided the foundation of prevention and treatment of toxoplasmosis.