人源抗乙型肝炎病毒表面抗原基因工程IgG全抗体的表达、纯化及初步鉴定  

Expression,Purification and Preliminary Identification of Recombinant Human Complete IgG Antibody against HBsAg

作　　者：黄仕和[1] 周志军[1] 彭祥兵[1] 詹骞[1] 张爱华[1] 闭兰[1] 余模松[1] 梁米芳[2] 

机构地区：[1]武汉生物制品研究所,武汉430060 [2]中国疾病预防控制中心病毒病控制所,北京100052

出　　处：《中国生物制品学杂志》2007年第12期873-876,共4页Chinese Journal of Biologicals

摘　　要：目的对人源抗乙型肝炎病毒表面抗原的基因工程IgG全抗体进行表达、纯化及初步鉴定。方法用含Fc片段抗-HBsAg Fab抗体基因的载体pAC-HBs-Fc,与杆状病毒线性DNA共转染昆虫细胞sf9,产生重组抗-HBsAg的全抗体。以不同浓度的HBsAg包被酶标板孔,用间接ELISA法检测培养上清中抗体的表达及特异性;用蛋白G亲和层析柱进行抗体的纯化,并对纯化的抗体进行SDS-PAGE、Western blot和竞争性ELISA分析。结果上清中表达的重组IgG抗体仅与HBsAg呈阳性反应,特异性良好,纯化后其纯度达97.1%。经SDS-PAGE和Western blot分析可见,IgG抗体轻链和重链的相对分子质量分别约为27000和55000,为人源IgG抗体。CHO表达的HBsAg和血源性HBsAg能竞争性抑制该重组IgG抗体与E.coli表达的HBsAg反应,其抑制率分别为55.9%和81.9%。结论人源抗乙型肝炎病毒表面抗原的基因工程IgG全抗体可在杆状病毒载体表达系统中成功表达。Objective To express, purify and preliminarily identify recombinant human complete IgG antibody against HBsAg. Methods Comnsfect st9 insect cells with recombinant plasmid pAC-HBs-Fc containing Fc fragment anti-HBsAg Fab antibody gene and the linear DNA of baculovirns for expression of recombinant hmnan complete IgG antibody against HBsAg.The expressed product was analyzed for Slificity by indirect ELISA using the microtiter plate coated with HBsAg,then purified by protein G affinity chromatography and identified by SDS-PAGE,Western blot and competitive ELISA.Results The expressed recombinant human complete IgG antibody was highly specific to HBsAg and reached a purity of 97.1% after purification. SDS-PAGE and Western blot proved the expressed product as human complete IgG antibody, of which the relative molecular masses of light and heavy chains were about 27 000 and about 55 000 respectively. The HBsAg expressed in CHO cells and plasma-derived HBsAg competitively inhibited the reaction of expressed recombinant human complete IgG antibody with HBsAg expressed in E. coli,with inhibition rates of 55.9% and 81.9% respectively. Conehrsion Recombinant human complete IgG antibody against HBsAg was successfully expressed in baculovirus expression system.

关 键 词：乙型肝炎病毒表面抗原 IgG 基因工程抗体 杆状病毒 表达 纯化 

分 类 号：Q786[生物学—分子生物学]