重组PTD-鳌虾凝集素抗对虾WSSV免疫机理研究

White spot syndrome virus is a highly pathogenic virus that infects crayfish and other crustaceans. C-type lectins may function as pattern-recognition receptors (PRRs) and play important roles in innate immune responses. Cell-penetrating peptides are short peptides that facilitate cellular uptake of various molecular cargoes, and one well known example is TAT peptide from HIV-1 TAT protein,based on this we creat of novel protein transduction domain (PTD) mutants.In this study, recombinant plasmids pET-28a(+)-PTD-PcLT are constructed and transformed into Escherichia coli strain to express PTD-PcLT fusion proteins. Elisa is used to confirm that PTD fusion proteins can translocate from the intestine to haemolymph of the crayfish. After immunization, cellular immunity(phagocytosis、encapsulation) and humoral immunological parameters (POD、SOD)are analyzed by FCM 、qRT-PCR and biochemical tests ,it is validated that PTD-PcLT is involved in the innate immune response of shrimp .To further confirm that recombinant PTD-PcLT can interact with WSSV, dot blotting and pull-down assay are performed. Shrimp are vaccinated by oral administration of rPTD- PcLT, following challenge with WSSV after the last day of feeding, calculating relative percent survival (RPS) . The research reveal the potential signal transduction pathways activated by C-type lectins and molecular mechanism against WSSV in invertebrate immunity .It provides a theory for anti-WSSV by oral lectin immune immunomodulator and high application value for herd immunity of shrimp.

白斑综合症病毒(WSSV) 是引起对虾急性爆发性疾病的病原体。虾凝集素（PcLT）是先天性免疫中抗病原微生物重要组成部分。穿膜肽PTD是一种能介导外源物质跨膜的小分子多肽。本研究拟在E.coli系统构建PTD-鳌虾PcLT的工程菌株；采用Elisa验证PTD- PcLT穿肠功能；通过qRT-PCR、生化技术检测PTD-PcLT对对虾多种体液免疫因子mRNA转录水平或酶活影响;采用FCM检测PTD-PcLT对细胞免疫（吞噬、包封）的影响；采用dotblotting验证PTD-PcLT与WSSV结构蛋白的结合作用；通过口服PTD-PcLT验证抗WSSV的作用。该研究对揭示PTD-PcLT激活下游信号通路及抗WSSV分子机制具有重要理论意义。为口服PTD-PcLT免疫调节剂抗WSSV提供理论依据，对实现虾群体免疫具有重要应用价值。

白斑综合征病毒（WSSV）是引起对虾急性爆发性疾病的病原体。虾凝集素（PcLT）是先天性免疫中抗病原微生物重要组成部分。穿膜肽(PTD)是一种能够接到外源物质跨膜的小分子多肽。本研究在E.coli系统构建PTD-螯虾PcLT的基因工程表达菌株；采用FCM、IF等方法验证PTD-PcLT穿肠功能；通过qRT-PCR、生化技术检测PTD-PcLT对螯虾多种体液免疫因子（peroxinectin、PO、SOD）mRNA转录水平或酶活的影响；Dot blot和pull-down检测PTD-PcLT结合WSSV病毒粒子的能力。该研究揭示了PTD-PcLT激活螯虾下游信号通路及PTD携带蛋白进入虾体内的作用。为口服PTD-PcLT免疫调剂抗WSSV提供理论依据，对实现虾群体免疫具有重要价值。