| Toxoplasma gondii is an obligate intracellular parasite, belonging to the apicomplexan protozoa. T. gondii has a complex life cycle, which needs intermediate host and terminal host. The intermediate host includes a variety of warm blooded vertebrates such as human, birds and fish. The terminal host is cat. It’s fatal that immunosuppressed individuals or immune disorder population are infected by this patogen birth. Maternal infection during pregnancy will cause miscarriage, stillbirth or premature. Miscarriage is also caused in female animals. Currently, toxoplasmosis has been a serious threat to human, animal health, and food security, and T. gondii typeⅡis one of mainly infected type of humans and animals. However, there is still no effective drugs or vaccines to prevent or treat this disease. Therefore,understanding the mechanism of T. gondii invasion to the intestinal, and looking for effective antigen molecules are important research direction to prevent and control toxoplasmosis.In order to understand the mechanism of T. gondii type Ⅱ invasion to mice intestinal cel, oocytes were used to infect mice, and intestinal secretory proteins were isolated after 6 hours post infection. The proteins were identified by mass and analyzed with bioinformatics’ soft. The results showed that 71 secreted proteins were obtained. The function of these proteins were mainly embodied as binding and catalysis, which involved in cell processes and metabolic processes, and compositions of cells and organelles.In order to know the differential gene expression of T. gondii in acute infection phase, T. gondii cysts were used to infect mice and total RNAs of brain tissues of mice on 10 days post infection were extracted for transcriptome high-throughput sequencing. The results showed that compared with the control group, 14506 new transcripts were detected, in which there were 12247 belonging to a novel splicing isoforms as known proteins, 315 proteins belonging to new genes, and other 1944 genes belonging to long noncoding RNA. Total 454 differentially expressed genes were found, in them there were 416 up-regulated expressing genes and 38down-regulated expressing genes. They involved in molecular or cell composition,binding, biological regulation, cell process etc.. Compared with T. gondii database,154 differentially expressing protein were obtained.To clone and express an identified secret protein code gene, T. gondii serine/threonine protein phosphatases 1(TgPP1) gene, a pair of specific primers were designed and used to amplify the gene by PCR with cDNA of T. gondii type Ⅱ cysts.The amplified product was then cloned into pMD18-T vector. The recombinant plasmids were digested with double enzymes and the target fragment was connected to the pET30 a vector for prokaryotic expression. The reactionogenicity of purified expressed product was detected by western blot. The results showed that tgpp1 gene was successfully amplified and the size was 921 bp similar to the size expected.SDS-PAGE analysis showed that recombinant Tg PP1(rTgPP1) protein was expressed successfully in soluble type and the molecular weight was approximately 33 kDa.Western blot analysis demonstrared that the purified expressed protein can be recognized by positive serum from immunized mice indicating that the rTgPP1 protein has a good reactionogenicity.The study successfully analyzed the secreted proteins and transcriptome at the invasion period of T. gondii typeⅡ, and the code gene of identified secret protein, T.gondii serine/threonine protein phosphatase 1 gene was successfully cloned and expressed. These reasults benefit to understand the infected and pathogenic mechanisms, develop new preventing and control strategy and reduce the spread of toxoplasmosis in human being and animals. |
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