Comparative Proteomics Analysis Of Three Developmental Stages Of Trichinella Spiralis

Trichinella spiralis, an intracellular parasite nematode, can cause severe foodborne parasitic zoonosis named Trichinellosis. Life cycle of T. spiralis consist of adult (Ad), muscle larvae (ML) and newborn larvae (NBL). The adult locates in the intestinal epithelial cells and ML lives in the skeleton muscle cells. In order to adapt to the parasitism, proteins of T. Spiralis expressed in different developmental stages are different. Studying stage-specific proteins is of significance to illustrate biological problems such as regulation of development and host-parasite interaction.In this study, iTRAQ was applied to identify proteins expressed in three developmental stages of T. Spiralis and a total of4691proteins were identified. Comparing those differentially expressed proteins separately, proteins which were important in immune regulation and parasite invasion were discovered. GO analysis discover that the identified proteins were mainly involved in "cell process and metabolic process","cell and cell component","binding and catalytic activity". Pathway enrichment showed that differentially expressed proteins were mainly involved in metabolic pathway, cell process and biology system pathway. Quantitative PCR was used to test transcriptional level of some genes from the three stages and the results showed little difference with that from iTRAQ, which demonstrated the reliability of iTRAQ analysis.Three differentially expressed T. spiralis thioredoxin peroxidase (TsTPxl, TsTPx2, TsTPx3) were selected from results of proteomics analysis. Specific primers of TsTPx1, TsTPx2, TsTPx3were designed from genomic and EST sequences and RT-PCR was applied to amplify the full ORF of TsTPx1, TsTPx2, TsTPx3. The sequences analysis indicated that recombinant TsTPx1, TsTPx2, TsTPx3proteins, having a AhpC-TSA domain and a1-cysPrx_C domain, belong to typical2-Cys Prx family. Quantitative PCR results showed that TsTPx1, TsTPx2, TsTPx3expressed in all three stages and TsTPx1was higher in ML, TsTPx2and TsTPx3were higher in Ad3. TsTPx1, TsTPx2, TsTPx3genes fragments were cloned into pET30a vector and recombinant pET30a-TsTPx1, pET30a-TsTPx2, pET30a-TsTPx3were transformed into BL21(DE3) to induce expression. Results showed Recombinant TsTPx1, TsTPx2, TsTPx3proteins were highly expressed with33.8ku,27.7ku,28ku respectively and Recombinant TsTPxl protein existed as insoluble body, while recombinant TsTPx2, TsTPx3proteins existed in soluble. Western-blot indicated that recombinant TsTPx1protein can be recognized by T. spiralis infected swine serum whereas recombinant TsTPx2, TsTPx3proteins cannot be recognized by the same serum. Enzyme catalytic experiments showed recombinant TsTPx1, TsTPx2, TsTPx3proteins have the ability to reduce H2O2and the ability increased with increasing protein concentration and growing time. This will lay the foundation for further study of function of thioredoxin peroxidase of T. Spiralis.