泛影葡胺密度梯度离心法纯化近江牡蛎类立克次体  

Purification of Rickettsia-like Organisms from Oyster,Crassostrea ariakensis by Renografin Density Gradient Centrifugation

作　　者：孙敬锋[1] 吴信忠[2] 

机构地区：[1]天津农学院水产科学系天津市水产生态及养殖重点实验室 [2]浙江大学动物科学学院,杭州310029

出　　处：《天津农学院学报》2008年第2期1-6,共6页Journal of Tianjin Agricultural University

基　　金：国家自然科学基金项目"海水养殖贝类立克次体病原蛋白抗原特性研究"(39970581);中国博士后科学基金项目"海洋贝类中类立克次体病原分子分类地位的鉴定"(20060390731);广东省自然科学基金项目"海水养殖贝类中类立克次体病原系统分类地位的鉴定"(06300897)

摘　　要：用国产泛影葡胺做介质,通过差速离心和密度梯度离心从患病近江牡蛎的鳃、外套膜、肝胰腺组织中纯化类立克次体。纯化的类立克次体在透射电镜下观察,20%～25%和25%～30%之间的条带为纯净的类立克次体,大多数呈球形、椭球形,偶见哑铃状(处于二分裂阶段)。纯化的类立克次体细胞形态完整,在光滑的细胞壁外围有一层疏松的粘液层,而且多个类立克次体常聚集在一起。利用API ZYM半定量法对类立克次体进行酶谱分析,检测到了11种酶,包括碱性磷酸酶、酸性磷酸酶、酯酶(C4)、类脂酯酶(C8)、亮氨酸芳胺酶、萘酚AS-BI-磷酸水解酶、β-半乳糖苷酶、N-乙酰葡萄糖胺酶、β-果糖酶、β-糖醛酸苷酶和缬氨酸芳胺酶。本实验证明,国产泛影葡胺是直接从患病贝类组织中纯化类立克次体的良好介质。The rickettsia-like organism （RLO） was purified by the use of differential speed centrifugation and renografin density gradient centrifugation directly from the gill, mantle, and digestive gland of the infected oyster, Crassostrea ariakensis. The purity and integrity of purified prokaryotes were validated by transmission electron microscopy. After renografin density gradient centrifugation, two minor bands at the 20%-25% interface or 25%-30% interface contained pure RLOs. Most organisms were often round, or ellipse but dumb bell occasionally （tmdergoing binary fission）. Their cell walls were untouched, and sometimes the obvious slime layer around them were observed, though the RLOs often gathered together. The enzymatic characteristics of the purified RLOs were studied. A total of 11 enzymatic activities, such as alkaline phosphatase, esterase （C4） , esterase lipase （C8） , leucine arylamidase, valine arylamidase, acid phosphatase, naphtol-AS-BI-phosphohidrolase, β-galactosidase, β-glucuronidase, N-acetyl-β-glucosaminidase and a-fucosidase were detected from purified RLOs. These conclusions show that renografin for the density gradient centrifugation was the good medium to purify the RLO from infected tissues of the mollusk.

关 键 词：泛影葡胺 类立克次体 纯化 近江牡蛎 酶谱分析 

分 类 号：S917.1[农业科学—水产科学]