目的 筛选和鉴定与东方田鼠抗性基因整合素连接激酶相关丝氨酸/苏氨酸磷酸酶（ILKAP）表达蛋白相互结合的日本血吸虫童虫蛋白。 方法 提取东方田鼠骨髓细胞蛋白和日本血吸虫童虫蛋白,进行免疫共沉淀,实验组为东方田鼠骨髓蛋白+日本血吸虫童虫蛋白+ILKAP抗体+磁性珠子,阴性对照组以兔IgG抗体代替ILKAP抗体,阳性对照组为东方田鼠骨髓蛋白或日本血吸虫童虫蛋白。十二烷基磺酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）对免疫沉淀复合物进行分离,切取差异性蛋白条带通过基质辅助激光解吸飞行时间质谱仪（MALDI-TOF-MS）鉴定,获得的混合物肽片段利用Mascot软件进行分析检索。分别以东方田鼠骨髓cDNA和日本血吸虫童虫cDNA为模板,RT-PCR扩增东方田鼠ILKAP（MfILKAP）基因和日本血吸虫表膜抗原（SjTA）基因。将MfILKAP和SjTA基因分别克隆至真核表达载体Flag-pCMV-2b和Myc-pcDNA3.1/(-)b中,构建的重组质粒Flag-ILKAP-pCMV-2b和Myc-TA-pcDNA3.1/(-)b共转染至HEK293T细胞中作为实验组,设阴性对照组Myc-TA-pcDNA3.1/(-)b或Flag-ILKAP-pCMV-2b和空白对照组Myc-pcDNA3.1/(-)b或Flag-pCMV-2b。用抗体Myc、Flag以及ILKAP分别与转染后的HEK293T细胞总蛋白进行免疫共沉淀和蛋白质印迹（Western blotting）分析。 结果 东方田鼠骨髓细胞蛋白和日本血吸虫童虫蛋白经过免疫共沉淀实验,结果显示,实验组与阴性对照组、阳性对照组相比,有差异条带。经MAIDI-TOF-MS鉴定,利用Mascot软件中的串级质谱数据搜索功能进行搜索鉴定,鉴定出与东方田鼠ILKAP抗体相互作用的日本血吸虫童虫蛋白8种。RT-PCR结果显示,ILKAP和TA在东方田鼠骨髓和日本血吸虫童虫中均有表达,东方田鼠骨髓ILKAP的扩增产物为1 100~1 200 bp,理论值为1 147 bp,日本血吸虫童虫TA基因的扩增产物大小为500~600 bp,理论值为561 bp。质粒pCMV-Tag 2b、pcDNA3.1/myc-His(-)b经酶切后,均有单一条带,分别约为4 300、5 500 bp;重组质粒Flag-ILKAP-pCMV-2b经酶切鉴定后,其大小分别为4 300和1 147 bp,Myc-TA-pcDNA3.1/(-)b经酶切鉴定后,其大小分别为5 500和561 bp。Western blotting分析结果显示,两种重组真核表达载体共转染实验组的PVDF膜分别与Myc、Flag以及ILKAP抗体孵育后有条带出现,而其他对照组无条带出现。 结论 鉴定出与MfILKAP抗体相互作用的日本血吸虫童虫蛋白8种,在HEK293T细胞中共表达了与MfILKAP结合的SjTA基因,MfILKAP与SjTA之间存在直接相互作用。

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.