旋毛虫免疫抑制分子——丝氨酸蛋白酶TspE1的酶学特性鉴定及其分子作用机制研究

The proteases from excretory/secretory products (ESP) of the nematode play an important role in invasion and interaction with the host. From our recent research results, the serine protease TspE1 which was released from Ad3 stage inhibits macrophage proliferation and the immune protection of the protein. The inhibitory effect of TspE1 can be abolished by serpin or anti-rTspE1 antibody treatment. Similarly, the inhibition of macrophage proliferation by ESP of T. spiralis Ad3 could also be inhibited by the anti-rTspE1 antibody. By analysing the gene sequence of TspE1, it is shown that TspE1 does not possess the topic serine protease reaction centre and active site which already known. We speculated that this is a novel protease that has not yet been discovered as an enzyme involved in the interaction between the Trichinella spiralis pathogen and the host. In this project, We focus on analyze the TspE1 X-ray crystal structure to confirm the active center with specific amino acid. We identify the protein molecules interacting with macrophages by CO-immunoprecipitation and point mutation-inactivated of TspE1. HPLC-MS and N-terminal sequencing determined the TspE1 substrate molecular cleavage sites. Through the analysis of the substrate molecular signaling pathway in macrophages and the knockout verification of the corresponding genes, the molecular mechanism of TspE1 inhibiting macrophage proliferation was investigated. In order to elucidate the molecular characteristics of TspE1 enzymatic properties and interaction with the host; furthermore reveal the possible immune evasion mechanism of Trichinella It is expected to provide a scientific basis for the prevention, diagnosis and treatment of trichinosis.

旋毛虫排泄分泌物（ESP）中蛋白酶类在其入侵中发挥重要作用。我们最新研究发现，旋毛虫感染后第3天成虫分泌一种明显抑制宿主巨噬细胞增殖且具有丝氨酸蛋白酶活性的蛋白（TspE1），其活性既受丝氨酸蛋白酶抑制剂抑制又可被旋毛虫感染血清阻断。但序列比对及结构分析显示该分子并不具有已知丝氨酸蛋白酶结构域及关键活性氨基酸，推测其可能为一种（类）参与病原与宿主互作的新型丝氨酸蛋白酶。本项目拟对TspE1晶体结构进行解析，鉴定其酶活结构域及关键活性氨基酸；利用点突变失活TspE1,免疫共沉淀鉴定出巨噬细胞中其互作蛋白；利用高效液质联用及N末端测序鉴定TspE1在底物分子上识别序列及切割位点；通过巨噬细胞中底物分子信号通路分析及相应基因敲除，探明TspE1抑制巨噬细胞增殖的分子机制。明确该新型丝氨酸蛋白酶分子特性，为旋毛虫引发宿主免疫抑制分子机制的阐明奠定基础，可望为旋毛虫病的诊、防、治提供可能的分子靶标。

旋毛虫病是由人或动物感染旋毛虫而引发的一种具严重危害性的人兽共患寄生虫病，旋毛虫所分泌的丝氨酸蛋白酶可以进入宿主并诱导宿主产生免疫反应，虫体所分泌的丝氨酸蛋白酶TspE1通过和宿主相互作用进而影响其侵染和寄生。项目实施中1）我们通过生物信息学方法对TspE1进行分析，同时对其结构进行模拟解析。2）我们利用不同丝氨酸蛋白酶底物对TspE1进行活性检测，进一步明确其理化特性。3）通过研究其对巨噬细胞的抑制作用及其互作机制，探究其免疫作用机制。4）利用包含TspE1的ES制备的抗原，进一步建立了旋毛虫病快速检测新的方法。综上所述，我们对TspE1从酶学特性，理化性质和免疫互作机制及其相关蛋白的检测进行了深入研究，其结果为旋毛虫的防治和诊断奠定了理论基础和应用前景。