猪带绦虫六钩蚴45W-4BX抗原的制备及其杂交瘤细胞株的建立  

Preparation of 45W-4BX antigen of Taenia solium oncosphere and establishment of its hybridoma cell lines

作　　者：孙晓林[1] 骆学农[1] 景志忠[1] 王佩雅[1] 才学鹏[1] 

机构地区：[1]中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室甘肃省动物寄生虫病重点实验室

出　　处：《中国兽医科学》2008年第2期91-96,共6页Chinese Veterinary Science

基　　金：国家“十一五”高技术研究发展计划(863)项目(2006AA10A207)

摘　　要：为获得针对猪带绦虫六钩蚴(TSO)45W-4BX抗原的杂交瘤细胞株,用IPTG对TSO45W-4BX的重组质粒pGEX-4BX进行了诱导表达,并采用Sepharose-4B层析技术对表达产物进行纯化,运用SDS-PAGE和Western-blot分别对其纯度和活性进行了检测。分别用弗氏完全佐剂、弗氏不完全佐剂乳化已纯化的猪带绦虫TSO45W-4BX重组抗原,随后用其分别免疫BALB/c小鼠3次,检测血清抗体效价呈阳性。之后,再进行加强免疫,取免疫小鼠脾细胞和SP2/0细胞进行融合,经TSO45W-4BX抗原和GST抗原双系统间接ELISA筛选,最终获得了18株抗TSO45W-4BX抗原的杂交瘤细胞株。采用有限稀释法对其中的4株进行5次亚克隆,最终获取了4株稳定分泌针对TSO45W-4BX抗原的单克隆抗体的杂交瘤细胞株,金标试纸条法鉴定其抗体亚类均属于IgG1类,轻链为κ型。To obtain the hybridoma cell line specific to 45W-4BX antigen of Taenia solium oncosphere （TSO）, the recombinant plasmid pGEX-4BX containing TSO45W-4BX gene was highly expressed in Escherichia coli induced with IPTG. The purity and activity of the expressed recombinant protein TSO45W-4BX purified by the Sepharose-4B chromatographic technique were analyzed by SDS-PAGE and Western-blot, respectively. BALB/c mice were immunized with the purified TSO45W-4BX antigen emulisified with Ferund＇s complete and incomplete adjuvant respectively. After three immunizations and the booster with the purified TSO45W-4BX protein,the SP2/0 cells were fused with splenocytes from the immunized mice. After screening with TSO45W-4BX antigen and GST protein by indirect ELISA, 18 positive hybridoma cells lines were obtained. Four of them were further sub-cloned for five times by the limited dilution method and eventually four monoclonal cell lines were obtained. The antibodies identified by the colloidal gold labeled paper represented IgG1 subclass,and their light chains represented κ type.

关 键 词：猪带绦虫 六钩蚴 TS045W-4BX抗原 杂交瘤细胞 

分 类 号：S852.734[农业科学—基础兽医学] Q78[农业科学—兽医学]