乙型肝炎病毒L蛋白颗粒的真核表达与纯化
乙型肝炎病毒;COS-7细胞系;重组质粒;超速离心;酶联免疫吸附试验;L蛋白;免疫印迹;透射电镜,曾建平,易继林,杨志芳,通讯作者:,ExpressionandpurificationoflargehepatitisBvirussurfaceantig
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曾建平, 中国人民解放军总医院肝胆外科 北京市 100853
易继林, 杨志芳, 华中科技大学同济医学院附属同济医院普通外科 湖北省武汉市 430030
通讯作者: 曾建平, 100853, 北京市, 中国人民解放军总医院肝胆外科. zjpdoc@163.com
电话: 010-66937781
收稿日期: 2006-12-10 接受日期: 2007-01-10
Expression and purification of large hepatitis B virus surface antigen particles produced in eukaryotic cells
Jian-Ping Zeng, Ji-Lin Yi, Zhi-Fang Yang
Jian-Ping Zeng,Department of Hepatobiliary Surgery; General Hospital of Chinese PLA, Beijing 100853, China
Ji-Lin Yi, Zhi-Fang Yang,Department of General Surgery, the Affiliated Tongji Hospital of Tongji Medical College of Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, China
Correspondence to:Jian-Ping Zeng, Department of Hepatobiliary Surgery; General Hospital of Chinese PLA, Beijing 100853, China. zjpdoc@163.com
Received:2006-12-10 Accepted:2007-01-10
Abstract
AIM: To explore the expression and purification of hepatitis B virus L particles in eukaryotic cells and to analyze their physical properties and antigenicity.
METHODS: COS-7 cell lines were transiently transfected with recombinant eukaryotic expression vector pSVsigLM-S-. The products were purified from cell culture supernatants after 48 h culture by a combination of CsCl isopycnic ultracentrifugation, density gradient ultracentrifugation in sucrose, and enzyme-linked immunosorbent assay (ELISA). Molecular sizes and antigenicity of the purified protein were determined by SDS–PAGE and Western blotting, respectively. The physical properties of samples were characterized by transmission electron microscopy (TEM).
RESULTS: L particles were successfully purified from transfected COS-7 cell medium by ultracentrifugation combined with ELISA. Cscl isopycnic ultracentrifugation showed that the area where density was 1.21 g/cm3 was rich in S-antigenicity. L particles exclusively consisted of glycosylated 42 kDa proteins that had S and PreS1 antigenicity ......
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