60000SSA抗原的提取及纯化  

Extraction and purification of 60 000 SSA antigen

作　　者：徐东[1] 张奉春[1] 甘晓丹[1] 唐福林[1] 董怡[1] 

机构地区：[1]中国医学科学院中国协和医科大学北京协和医院风湿免疫科,100730

出　　处：《中华风湿病学杂志》2003年第1期10-13,共4页Chinese Journal of Rheumatology

基　　金：国家自然科学基金资助项目 (3 9970 698) ;首都医学发展科研基金资助项目 (首都ID19980 1)

摘　　要：目的　建立用于临床检验及研究的SSA 6 0 0 0 0抗原的提取及纯化方法。方法　利用牛脾组织提取粗抗原 ,采用阴离子交换层析法 (IEC)进行初纯化 ,再分别以分子筛及亲和层析法进一步提纯 ,并利用对流免疫电泳 (CIE)、免疫扩散 (ID)及免疫印迹 (IBT)法检测各纯化阶段提取液中的 6 0 0 0 0SSA抗原。结果　IEC(DE5 2 )初步提纯SSA抗原效佳。 0 15～ 0 35mol/LNaCl(溶于 0 0 1mol/L磷酸盐缓冲液pH 7 2中 )是最佳洗脱范围 ;分子筛方法 (SephadexG 10 0 )作为SSA抗原的进一步提纯方法不理想 ,杂蛋白存留较多。但可作为用于分析目的的初纯化方法 ;亲和层析方法可进一步纯化出 6 0 0 0 0SSA抗原。十二烷基硫酸钠 聚丙烯酰胺凝胶电泳 (SDS PAGE)转膜后氨基黑染色见只有一条条带位于 6 70 0 0～ 4 30 0 0。进一步行IBT证实其为 6 0 0 0 0SSA抗原。结论　利用牛脾粗抗原经IEC、亲和层析的方法提纯 6 0 0 0 0SSA抗原操作简便 ,纯化效果好 ,价格不高 ,能够成功地制备6 0 0 0 0SSA抗原 ,为开展 6 0 0 0 0SSA抗原在疾病中作用的研究创造了条件。Objective To establish extracting and purifying methods of 60 000 SSA antigen in order to apply cheap and good 60 000 SSA antigen for clinical tests and researches.Methods Calf spleen extract was prepared,using IEC (DE52),gel filtration chromatography (Sephadex G 100) and immunoadsorbent chromatography for purification of 60 000 SSA antigen.CIE,ID and IBT againse extracts after every purification step were done to detect 60 000 SSA antigen.Results IEC (DE52) was a good method for initial purification of SSA antigen.Elution from phosphate buffer 0 01 mol/L (pH 7 2) 0 15 mol/L NaCl gradient to 0 35 mol/L NaCl gradient was best for SSA antigen.Gel filtration chromatography (Sephadex G 100) was unfit for further purification of SSA antigen.But just as an initial purification method,it was feasible.Immunoadsorbent chromatography was one of the best methods for further purification of 60 000 SSA antigen.SDS PAGE stained with Coomassie blue showed a single polypeptide between 67 000 and 43 000 .Western blot confirmed it was 60 000 SSA antigen.Conclusion It is simple to prepare and operate immunoaffinity column,it is ideal to prepare 60 000 SSA antigen,which paves a way for studying the relation of 60 000 SSA antigen disease.

关 键 词：SSA抗原 提取 纯化 离子交换色谱法 凝胶色谱法 亲和色谱法 

分 类 号：R392.11[医药卫生—免疫学]