弧菌复苏促进因子基因结构、酶活性及其在非可培养状态细胞复苏过程中作用机制

Vibrios naturally inhabit marine and estuarine environments.Some Vibrio species are known to be the causative agent of human and animal diseases.It has been reported that many Vibrio species could enter into the viable but nonculturable (VBNC) state as a response to natural stresses.Under appropriate conditions these VBNC microorganisms can be able to recove from this dormant state,becoming metabolically active and fully culturable.However,the mechanisms of the resuscitation remains unknown.The resuscitation-promoting factor is a novel bacterial cytokine family, which is widely distributed in the most Gram-positive and some of Gram-negative bacteria. But the gene structure and distributions vary in different bacteria. The aims of the project are as follow: The genes of the resuscitation-promoting factor like proteins will be cloned and expressed from Vibrio species. The gene type, structure and distribution of the resuscitation-promoting factor like proteins in Vibrio species will be analyzed.The enzymatic activities will be studied. The active site of the proteins will be studied by PCR based site-direct mutation,and the relationship of activity of the enzyme with its molecular structure will be studied.The resuscitation-promoting factor mutant strains will be constructed to evaluate the roles of these proteins in resustation from the VBNC state.The expressed resuscitation-promoting factor like proteins will also be used for the culturation of uncultuable microorganisms. New ideas and methods for fish disease control will be developed.

弧菌广泛分布于河口及海洋环境，部分弧菌是人类和其他动物的重要病原菌。弧菌在不良环境形成非可培养的休眠状态，条件适宜时复苏为可培养形式，但其复苏机制仍不清楚。已发现许多细菌存在能促进休眠细胞复苏的细胞因子（Resuscitation-promoting factor, Rpf），且不同种类的细菌其复苏因子蛋白基因结构及种类存在较大差异。本项目拟在前期工作基础上，对弧菌细胞复苏因子蛋白家族基因种类、结构和分布进行研究，表达并纯化主要弧菌复苏促进因子蛋白，利用体外定点突变技术研究其活性中心氨基酸组成，并研究其酶学性质；将表达的具有生物活性的复苏促进因子用于难培养微生物细胞的培养及增殖；利用同源重组技术构建复苏促进因子基因缺失突变株，研究不同突变株的生长和复苏能力，探索其在弧菌非可培养状态细胞复苏过程中的作用机制，为揭示弧菌病爆发和流行规律提供理论依据，为防止弧菌病提供有效抗原和药物作用靶点。

利用弧菌基因组数据库，从不同弧菌基因组扩增出细胞复苏促进因子家族yeaZ基因，发现该类基因在主要弧菌类群普遍存在，序列相似性在67％- 92%，与大肠杆菌糖蛋白酶、沙门氏菌及耶耳森菌M22 肽酶YeaZ序列相似性为55-59%，与藤黄微球菌、结核分枝杆菌等革兰氏阳性细菌细胞复苏因子相似性为27% - 36%。将弧菌这类基因归为yeaZ基因。将yeaZ基因克隆至原核表达载体pET-28a（+）表达，SDS-PAGE 显示纯化蛋白30 kDa。添加YeaZ对非可培养状态哈维氏弧菌有明显促进作用。当哈维氏弧菌在非可培养状态保存120天后，只有添加重组YeaZ才能实现细胞复苏。重组YeaZ蛋白能明显促进干燥和高盐环境土壤可培养微生物数量和种类。. 用人工合成底物4-Methylumbelliferyl-β- D-N-N’-N” – triacety lchitotriose 检测到弧菌复苏因子具有微弱溶菌酶活性。对其可能酶活性中心位点氨基酸Thr-71和Asp-112进行了定点突变，突变体酶活性部分降低，其突变体仍具有复苏促进作用。重组YeaZ蛋白还表现出一定的蛋白酶活性。Western-blotting 检测发现正常生长哈维氏弧菌、副溶血弧菌有27 kDa特异蛋白，且VBNC 状态的副溶血弧菌有2 条蛋白带，分子量分别为27 kDa和54 kDa，推测其可能存在二聚体。构建了哈维氏弧菌YeaZ突变株，发现突变株在低温条件进入活的非可培养状态及复苏特征与野生株无明显差别，非可培养状态120天后添加重组复苏促进因子YeaZ可培养细菌数量为1.44×103 CFU/ mL。.研究发现复苏促进因子yeaZ在弧菌种类普遍存在。重组蛋白能明显促进非可培养状态细胞复苏，对极端环境土壤非可培养微生物有一定复苏作用。重组蛋白具有微弱的溶菌酶活性和蛋白酶活性。定点突变发现其溶菌酶活性与弧菌复苏作用没有直接关系，与革兰氏阳性细菌复苏因子rpf可能存在不同的作用机制。研究结果对了解弧菌在极端环境适应机制，促进环境中难培养微生物的复苏有较为重要的理论意义和应用价值。