关键词: 东方田鼠, 日本血吸虫, 整合素连接激酶相关丝氨酸/苏氨酸磷酸酶, 免疫共沉淀

Abstract:

Objective To understand the mechanism underlying the natural resistance to Schistosoma infection in Microtus fortis, the schistosomula proteins interacted with ILKAP(integrin-linked kinase-associated serine/threonine phosphatase), one of the resistant-related proteins in M. fortis, were screened and identified. Methods The crude proteins were extracted from S. japonicum schistosomula and those proteins specifically bound with M. fortis ILKAP(MfILKAP) were isolated and identified by immunoprecipitation using magnetic beads conjugated with anti-ILKAP IgG. Briefly, the beads conjugated with anti-ILKAP IgG were co-incubated with bone marrow proteins of M. fortis and S. japonicum schistosomula extracts, the proteins bound to the beads were extracted in SDS-loading buffer and separated by SDS-PAGE. The separated bands were cut from the gel and analyzed by MALDI-TOF-MS. The schistosomula proteins bound to ILKAP were identified by searching through genome database of S. japonicum. In order to further determine the binding activity, genes for one of the ILKAP-bound S. japonicum proteins, tegument antigen （TA), and MfILKAP were amplified and cloned into eukaryotic expression plasmids Flag-pCMV-2b and Myc-pcDNA3.1/(-)b, respectively. The recombinant plasmid DNAs were co-transfected into HEK293T cells. The expression of recombinant tegument antigen of S. japonicum (SjTA) and MfILKAP and their interaction were confirmed by co-immunoprecipitation with anti-ILKAP. Results A total of 8 S. japonicum schistosomula proteins bound to MfILKAP were identified by co-immunoprecipitation with anti-ILKAP IgG and MALDI-TOF-MS analysis. One of the major ILKAP-binding proteins was SjTA. After RT-PCR amplification and enzymatic digestion, fragments of 1 147 bp, and 4 300 bp from M. fortis, and fragments of 561 bp and 5 500 bp from Schistosomula were obtained, respectively. In order to further determine their interacting activity, MfILKAP and SjTA were successfully cloned from their corresponding cDNA and the recombinant proteins were effectively expressed in HEK293T cells determined by Western blotting with specific antibodies. The binding activity of recombinant MfILKAP to recombinant SjTA was further confirmed by co-immunoprecipitation with anti-ILKAP IgG from the co-expressed cell lysates and recognition by the specific antibodies. Conclusion Total 8 S. japonicum schistosomula proteins have been identified to bind to MfILKAP. One of the ILKAP-binding proteins, SjTA, was cloned and co-expressed in HEK293T cells. The interaction between SjTA and MfILKAP was further confirmed by the binding of both recombinant proteins.

Key words: Microtus fortis, Schistosomula, Integrin-linked kinase-associated serine/threonine phosphatase, Co-immunoprecipitation