多头绒泡菌核仁及其骨架中原肌球蛋白的免疫细胞化学鉴定  

IMMUNOCYTOCHEMICAL IDENTIFICATION OF TROPOMYOSIN IN NUCLEOLI AND NUCLEOLAR MATRIX OF PHYSARUM POLYCEPHALUM 

作　　者：焦明大[1] 曾宪录[1] 王晓光[2] 郝水[1] 

机构地区：[1]东北师范大学遗传与细胞研究所,长春130024 [2]长春师范学院生地系,长春130024

出　　处：《微生物学报》1999年第5期402-407,共6页Acta Microbiologica Sinica

基　　金：国家自然科学基金

摘　　要：分离多头绒泡菌（ ｐｈｙｓａｒｕｍ ｐｏｌｙｃｅｐｈａｌｕｍ） 细胞的核仁，先用ＤＮａｓｅ Ｉ消化，去除核仁内的ＤＮＡ；然后用０－２５ｍｏｌ／Ｌ（ＮＨ４）２ＳＯ４ 和２ ｍｏｌ／ＬＮａＣｌ 相继抽提去掉大部分蛋白质，制备成核仁骨架。ＳＤＳＰＡＧＥ 分析结果表明，核仁骨架中含有约２０ 种多肽，其中包括３７ｋＤ 左右与原肌球蛋白分子量相当的多肽。以兔抗原肌球蛋白抗体为一抗，ＦＩＴＣ 标记的羊抗兔ＩｇＧ 抗体为二抗的间接免疫荧光检测结果表明，核仁和核仁骨架样品都能发出明亮的荧光，而对照样品未见明亮的荧光。间接免疫斑点印迹检测结果进一步证明，在核仁骨架的蛋白质成分中存在原肌球蛋白。胶体金免疫电镜检测结果显示，标记原肌球蛋白抗体的标本上有较多的金颗粒，而对照组标本上只有极少的金颗粒。金颗粒在核仁中主要呈散在分布。Nucleoli were isolated from physarum polycephalum ,and nucleolar matrix was prepared by digesting the nucleoli respectively with DNase I,0.25mol/L (NH 4) 2SO 4 and 2mol/L NaCl to remove DNA and most proteins.SDS PAGE analysis indicated that there were about 20 polypeptides in nucleolar matrix component,including the 37kD polypeptide which was similar to tropomyosin in molecular weight.The result of indirect immunoflurescence treated with anti tropomyosin antibody and sheep anti rabbit IgG antibody labelled with FITC showed that bright flurescence was observed in the nucleoli and nucleolar matrix,but no bright flurescence in the controls.Indirect immunodotting detection further verified that tropomyosin existed in nucleolar matrix.Protein A colloidal gold immunoelectron microscopic study showed that there were many gold particles in the specimens labelled with tropomyosin antibody,and there were few gold particles found in the controls.Tropomyosin distributed dispersely in nucleoli.

关 键 词：多头绒泡菌 核仁 核仁骨架 原肌球蛋白 鉴定 

分 类 号：Q932[生物学—微生物学]