SARS-CoV抗原的纯化与鉴定  

Purification and identification of SARS-CoV antigen

作　　者：张松乐[1] 田光[1] 周华[1] 张良艳[1] 王东勇[1] 阳幼荣[1] 马丽娟[1] 王希良[1] 

机构地区：[1]军事医学科学院微生物流行病研究所,北京100071

出　　处：《免疫学杂志》2005年第3期244-247,共4页Immunological Journal

基　　金：国家"863"基金(2003AA208224);军队"十五"重大攻关课题(03F003-3)资助项目

摘　　要：目的纯化灭活SARS-CoV为研制马抗SARS-CoV免疫球蛋白提供抗原.方法将灭活的SARS-CoV悬液超滤浓缩,用Sepharose 4 Fast Flow分子筛和阴离子交换柱层析纯化,用高效液相色谱、SDS-PAGE和电镜观察测定抗原的纯度.结果将01、02和03三批SARS-CoV悬液分别进行11.2倍、12.5倍和25.0倍超滤浓缩,抗原的回收率依次为48.6%、46.3%和55.6%,凝胶过滤层析后抗原的回收率依次为81.6%、79.3%和86.0%.再经阴离子交换梯度层析进一步提高了抗原的纯度和浓度,蛋白的比活性由纯化前的0.11U/μg增加到1.44U/μg.电镜下观察到SARS-CoV抗原颗粒完整,呈圆形或椭圆形状,直径在80～120 nm之间.SDS-PAGE显示主要条带位于Mr43 000～66000之间,经质谱扫描分析确定该成分为Mr46 000.高效液相层析法分析抗原纯度为97.0%.马抗SARS-CoV免疫球蛋白中和抗体效价在1:6 400～1:12 800之间.结论本研究为制备高效价的马抗SARS-CoV免疫球蛋白奠定了基础.Objective To prepare the immunoglobulin of horse anti-SARS-CoV by inactivating and purifying the SARS-CoV antigen. Methods The inactivated SARS-CoV suspension was concentrated by ultrafiltration, then purified by gel chromatography and ion exchange chromatography. The purity of the antigen was assayed by high-performance liquid chromatography (HPLC) and SDS-PAGE. Results The purity and concentration of SARS-CoV antigen were improved with the methods of ultrafiltration, gel chromatography, and ion exchange chromatography. Under electron microscope, the particles of SARS-CoV antigen were round or ellipse, whose diameters were between 80-120 nm. SDS-PAGE assay showed that the main belts were located between M _r 43 000 and 66 000 . HPLC assay indicated that the purity of antigen was 97.0%. Conclusion The SARS-CoV antigen is produced successfully.

关 键 词：SARS-COV 超滤 纯化 

分 类 号：R392[医药卫生—免疫学]