sPLA2是花生四烯酸炎症通路的上游开关。由于现有的COX-2抑制剂类抗炎症药物（即NSAIDs）毒副作用强烈，因此寻找靶向sPLA2的药物具有重要的理论意义和应用价值。申请者前期通过生物信息学和相关实验，发现华游蛇解蛇毒蛋白"γ型sPLA2抑制剂（PLIγ）"具有潜在的抑制人sPLA2活性，是理想的抗炎症候选药。在此基础上，本项目拟开展如下研究：1）制备PLIγ单克隆抗体，通过免疫共沉淀、双向电泳和western blot技术，研究与PLIγ有相互作用的细胞蛋白质；2）构建PLIγ-pIRES2-EGFP真核表达载体，转染人THP-1细胞株，通过LPS诱导实验及炎症因子、细胞因子检测，研究PLIγ抗细胞炎症效果；3）复制人SAP和RA动物模型，评价PLIγ整体水平抗炎症作用。通过以上蛋白质相互作用，体外细胞实验和动物模型三个层次研究，系统地阐述华游蛇PLIγ抗炎症效果及作用机制。

Secreted phospholipase A2 is a master switch in the upstream of arachidonic acid inflammatory pathway. Considered the severe side-effects of COX-2 inhibitor drugs, namely NSAIDs, which are the maximum prescription drug, drug discovery targeting to sPLA2 is of great significance both in anti-inflammatory pharmacology and clinical application. By the results of bioinformatics analysis and experimental trial in our preliminary study, we found an antidotal protein that isolated from Sinonatrix percarinata known as "γ type sPLA2 inhibitor (PLIγ)"，has potential inhibitive activity against human sPLA2. In this project, we are going to continue our research in three major aspects, 1) prepare the monoclonal antibody of PLIγ. With the mAb, co-immune precipitation will be carried out to screen human proteins (mostly, the PLA2s) that binding with PLIγ. These proteins are then separated by two dimensional electrophoresis(2DE) and identified by MS, and finally verified by western blot. 2) Develop a human monocyte cell line known as THP-1 that hosting PLIγ gene by transfection of recombinant plasmid of PLIγ-pIRES2-EGFP. After treatment with lipopolysaccharide(LPS), the recombined THP-1 cells and normal cells will exhibit different inflammatory response. Pro-inflammatory factors and cell factors in these cells are then assayed to assess the protective effect of PLIγ in vitro cell. 3) Replicate human severe acute pancreatitics (SAP) and rheumatoid arthritis（RA） on Wistar rats. Anti-inflammatory activities are valued in vivo on the two animal model. When the above studies on protein interactive, cell trial in vitro and animal experiment are finished, the anti-inflammatory effect and its related mechanism will be comprehensively illustrated.