麻风分枝杆菌热休克蛋白65的表达、纯化及其多克隆抗血清的制备  

Expression and Purification of Heat Shock Protein 65(HSP65) of Mycobacterium leprae and Preparation of Anti-HSP65 Polyclonal Antiserum

作　　者：吴娟[1,2] 范小勇[2] 曲勍[2] 马辉[2] 许艳[2] 罗玉萍[1] Douglas B.Lowire 

机构地区：[1]南昌大学生命科学与食品工程学院,南昌330031 [2]复旦大学附属上海市公共卫生临床中心,上海201508

出　　处：《中国生物制品学杂志》2011年第7期757-760,共4页Chinese Journal of Biologicals

摘　　要：目的原核表达、纯化麻风分枝杆菌热休克蛋白65(Heat shock protein 65,HSP65),并制备其多克隆抗血清。方法以含hsp65基因的真核表达质粒pCMV4.65为模板,PCR扩增hsp65基因,克隆入pET-28a(+)载体,构建重组表达质粒pET-28a-hsp65,转化大肠杆菌BL21(DE3),IPTG诱导表达。表达产物经镍离子亲和层析柱纯化后免疫BALB/c小鼠,制备多克隆抗血清,Western blot分析重组蛋白的反应原性。结果重组表达质粒pET-28a-hsp65经双酶切及测序证实构建正确;表达的重组蛋白相对分子质量约65 000,表达量约占菌体总蛋白的30%,主要以包涵体形式表达;纯化的重组蛋白纯度可达95%,浓度约为1.0 mg/ml;制备的HSP65多克隆抗血清效价可达1∶12 800;重组蛋白具有良好的反应原性。结论已成功表达、纯化了麻风分枝杆菌重组HSP65蛋白,并制备了HSP65多克隆抗体,为进一步研究以HSP65为基础的亚单位疫苗和核酸疫苗奠定了基础。Objective To express the heat shock protein 65（HSP65） of Mycobacterium leprae in prokaryotic cells,purify the expressed product and prepare anti-HSP65 polyclonal antiserum.Methods The hsp65 gene was amplified by PCR using plasmid pCMV4.65 carrying the target gene as a template,and cloned into vector pET-28a（＋）.The constructed recombinant plasmid pET-28a-hsp65 was transformed to E.coli BL21（DE3） for expression under induction of IPTG.The expressed product was purified by nickel ion affinity chromatography and used for immunization of BALB/c mice to prepare antiserum.The reactogenicity of recombinant HSP65 was analyzed by Western blot.Results Both restriction analysis and sequencing proved that recombinant plasmid pET-28a-hsp65 was constructed correctly.The expressed recombinant HSP65,with a relative molecular mass of about 65 000,contained about 30% of total somatic protein and mainly existed in a form of inclusion body.The purity and concentration of purified recombinant HSP65 were 95% and about 1.0 mg/ml respectively.The prepared anti-HSP65 antiserum reached a titer of 1 ∶ 12 800,and showed high reactogenicity.Conclusion The recombinant HSP65 of M.leprae was successfully expressed and purified,and anti-HSP65 polyclonal antiserum was prepared,which laid a foundation of further study on HSP65-based subunit vaccine and DNA vaccine.

关 键 词：分枝杆菌 麻风 热休克蛋白65 基因表达 多克隆抗体 

分 类 号：R378.41[医药卫生—病原生物学] R392.33[医药卫生—基础医学]