曼氏血吸虫虫卵白细胞介素-4诱导因子的原核表达及多克隆抗体的制备  

Prokaryotic Expression of IL-4-inducing Principle of Schistosoma Eggs of Sc-histosoma mansoni and Preparation of Polyclonal Antibody

作　　者：赵娜[1] 李世杰[1,2] 徐晓芳[1] 吴威[1] 范大为[1] 宫鹏涛[1] 李建华[1] 张西臣[1] 

机构地区：[1]吉林大学畜牧兽医学院,长春130062 [2]东北农业大学生命科学学院,哈尔滨150030

出　　处：《中国生物制品学杂志》2011年第10期1140-1143,共4页Chinese Journal of Biologicals

基　　金：国家传染病防治重大专项(2008ZX10004-001-B);吉林大学研究生创新基金资助项目(20091033)

摘　　要：目的原核表达曼氏血吸虫虫卵白细胞介素-4诱导因子(IL-4-inducing principle of schistosoma eggs,IPSE),并制备小鼠多克隆抗体。方法人工合成IPSE基因,采用PCR技术克隆其成熟体开放阅读框,插入原核表达载体pET-28a中,构建重组表达质粒pET-28a-IPSE。转化E.coli BL21(DE3),IPTG诱导表达。表达产物经SDS-PAGE和Western blot分析后,应用组氨酸标签蛋白纯化柱纯化包涵体蛋白,免疫BALB/c鼠,制备多克隆抗体。结果克隆的IPSE基因与GenBank中登录的基因序列同源性为100%;重组表达质粒pET-28a-IPSE经PCR及双酶切鉴定证明构建正确;表达的重组蛋白相对分子质量约为16 000,以包涵体形式表达,表达量约占菌体总蛋白的30%,可与鼠抗His单抗特异性结合;用纯化的包涵体蛋白免疫BALB/c小鼠后,获得的特异性抗血清效价为2×10-4,该血清可与IPSE蛋白发生特异性反应。结论已原核表达、纯化了曼氏血吸虫IPSE蛋白,并制备了高滴度的特异鼠抗血清,为进一步从日本血吸虫中筛选相似蛋白及研究其功能奠定了基础。Objective To expression the IL-4-inducing principle of schistosoma eggs（IPSE） of Schistosoma mansoni in prokaryotic cells and prepare mouse polyclonal antibody.Methods IPSE gene was synthesized artificially,of which the open reading frame（ORF） was cloned by PCR and inserted into expression vector pET-28a.The constructed recombinant plasmid pET-28a-IPSE was transformed to E.coli BL21（DE3） for expression under induction of IPTG.The expressed protein was identified by SDS-PAGE and Western blot,then purified by His GraviTrap protein purification column chromatography and used for immunization of BALB / c mice to prepare polyclonal antibody.Results Sequencing result showed a homology of 100% of the cloned IPSE gene to that reported in GenBank.Both PCR and restriction analysis proved that recombinant plasmid pET-28a-IPSE was constructed correctly.The expressed recombinant protein in a form of inclusion body,with a relative molecular mass of about 16 000,contained about 30% of total somatic protein and showed specific binding to mouse anti-His monoclonal antibody.Specific antisera at a titer of 2 × 10-4 was obtained by immunizing BALB / c mice with the purified inclusion body,which showed specific reaction with IPSE protein.Conclusion The IPSE of Schistosoma mansoni was expressed in prokaryotic cells and purified,and mouse antisera at high titer was purified,which laid a foundation of screening the similar protein from Schistosoma mansoni and studying its function.

关 键 词：血吸虫 曼氏 虫卵白细胞介素-4诱导因子 原核细胞 基因表达 包涵体 多克隆抗体 

分 类 号：R378.24[医药卫生—病原生物学] R392-33[医药卫生—基础医学]