与大鼠Morris肝癌7777相关的染色质蛋白的免疫亲和层析分离与鉴定

 用5mol/L尿素,将大鼠Morris肝癌7777染色质解离为染色质非组蛋白 (UP组分)及染色质沉淀(UC组分)。UP(含90—95％非组蛋白)用免疫亲和层析(与大鼠Morris肝癌7777去组蛋白染色质抗体交联)分级,经2mol/L NaSCN及8mol/L尿素分部洗脱。将UP及UC,来自UP亲和层析的2mol/L NaSCN及8mol/L尿素洗脱组分同时进行SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)。以大鼠Morris肝癌7777去组蛋白染色质抗体作探针,进行免疫显迹(Immunoblot)测定。在UP部分出现二条阳性带,分子量为:200K及116K。UC部分有三条染色不很深的阳性带,分子量为200K,118K及91K。来自UP亲和层析的2mol/L NaSCN及8mol/L尿素洗脱部分分别有一条浓而清晰的阳性带,分子量分别为74K及83K。用酶联免疫吸附法(E1isa)测试从UP凝胶上切割下的阳性区带,其免疫特异性显著。

THE ISOLATION AND IDENTIFICATION OF RAT MORRIS HEPATOMA 7777-ASSOCIATED CHROMOSOMAL PROTEINS BY IMMUNO-AFFINITYCHROMATOGRAPHY

In order to isolate and identify a group of hepatoma-associated nonhistone chromosomal proteins(MW 42-200k daltons), rat Morris Hepatoma chromatin was dissociated in 5.0mol/L urea and then,subjected to immuno-affinity chroma tography, SDS-PAGE and immunoblot or the enzyme-linked immunosorbent assay. The UP fraction in the chromatin contains 90-95% of chromosomal non-histone proteins. Immunoblots with the antisera against Morris hepatoma 7777 dehistonized chromatin showed that two positive bands with MW 200K and 116K were found in UP fraction, and three weakly positive bands with MW 200, 118 and 91K were present in UC fraction. Upon fractionation of the UP fraction by immunoaffinity-chromatography on a column cross linked with hepatoma antibody, single bands of 74K and 83K protein positive to immunoblot were identified in 2mol/L NaSCN and 8mol/L urea eluted fractions respectively.