表面抗原（VSP）变异是贾第虫等寄生生物逃避宿主免疫应答的主要策略，其机制研究对于阐明抗原变异的生物学功能和有效预防策略,具有重要意义。申请人前期工作已建立PCR为基础的特异性VSPs编码正义和反义mRNA的扩增技术，报道了贾第虫VSPs反义mRNA的转录，及在贾第虫VSPs表达后转录调控（PTGS）的作用。在此基础之上，本课题拟以VSP16单克隆贾第虫虫株作为研究对象，采用单克隆抗体免疫荧光技术（IFA）对VSP的变异进行监测；并通过对核初始mRNA转录分析及转录后胞浆反义mRNA的检测，阐明反义mRNA转录是VSPs变异的的重要环节；同时应用基因敲除技术，研究在VSPs转换过程中，RdRp对反义mRNA转录作用，及 Dicer对双链mRNA的切割作用，阐明以反义mRNA介导的贾第虫VSPs变异PTGS机制,为揭示VSPs变异机制以及生物体内反义mRNA的转录现象奠定生物学基础。

The variation of surface antigen is one of the major strategies that Giardia parasite to evade host immune attach. Further understanding the mechanism underlying the regulation of variant surface protein (VSP) during Gardia’s life cycle and parasitism would be very important for the control of the infection and treatment of giardiasis. In our previous study we have established the methods to identify the sense and anti-sense mRNAs for VSPs by PCR, the transcription of the anti-sense mRNA of VSPs and their possible post-transcriptional gene silencing (PTGS) effects on the regulation of VSP expression. In this proposal, we would like to target VSP16, one of the VSPs we identified in our previous study, as a model to further understand the mechanism behind the regulation of VSP. Through the expression of VSP16 recombinant protein, making the monoclonal antibody against VSP16, the variation of VSP16 expression will be monitored using immunofluorescence assay. At the same time, the transcription of VSP16 mRNA, sense and anti-sense mRNA of VSP16 will be investigated and the relationship between the expression of VSP16 on the surface of Giardia and the level of anti-sense mRNA will be analyzed. In order to understand the effects of RNA-dependent RNA polymerase (RdRP) and Dicer on the post-translational regulation of anti-sense VSP16 mRNA, the Duplex-specific nuclease (DSN) will be used to make miRNA to knock-out these two enzymes to identify their function on the regulation of VSP variant expression of Giardia. The understanding of the mechanism for the regulation of VSP expression will provide a pivot approach for the control of giardiasis that affect millions people in the world.