抗日本血吸虫单链抗体免疫毒素的构建 表达及生物学活性鉴定  

Construction,expression and identification of a recombinant immunotoxin of scFv against Schistosoma japonicum

作　　者：李红[1] 朱晓娟[2] 顾春艳[1] 任勇亚[1] 许静[1] 王忠灿[3] 李玉华[1] 仇镇宁[1] 朱进[3] 冯振卿[1] 管晓虹[1] 

机构地区：[1]南京医科大学卫生部抗体技术重点实验室,南京210029 [2]南京医科大学第二附属医院消化医学中心 [3]南京军区军事医学研究所

出　　处：《中国血吸虫病防治杂志》2010年第2期117-121,共5页Chinese Journal of Schistosomiasis Control

基　　金：国家高技术研究发展计划(863计划)(2006AA02Z415)

摘　　要：目的构建抗日本血吸虫单克隆抗体NP11-4单链抗体和绿脓杆菌外毒素衍生物PE38KDEL的原核融合表达载体,并对其表达产物进行生物学活性鉴定。方法应用PCR方法体外扩增NP11-4VH、VL、scFv和PE38KDEL基因,经测序后将scFv及PE38KDEL基因通过限制性内切酶双酶切及连接反应,构建原核表达载体pBAD/gIIIA-scFv-PE38KDEL,转化E.coliTop10F′,L-阿拉伯糖诱导表达,表达产物经Western blot及ELISA鉴定其活性。结果构建了重组免疫毒素表达载体pBAD/gIIIA-scFv-PE38KDEL,诱导表达产物主要以包涵体形式存在,分子量约为70kD;经变性、复性后的重组免疫毒素与血吸虫可溶性虫卵抗原有较好的结合活性。结论构建及表达了抗日本血吸虫单链抗体免疫毒素,为血吸虫病治疗提供了一条新思路。Objective To construct a recombinant immunotoxin expression vector composed of a single-chain Fv fragment of Schistosoma japomicum and PE38KDEL gene,and identify the binding activity of the purified product with SEA antigen.Methods The VH and VL genes were amplified by PCR from the parent monoclonal antibody NP11-4.Then the amplified scFv and PE38KDEL genes were inserted into the expression vector pBAD/gIII A.The fusion protein expressed in E.coli Top10F′ induced by L-arabinose.After purification,the activity of the immunotoxin was evaluated by Western-blot and ELISA.Results The new recombinant immunotoxin expression vector pBAD/gIII A-scfv-PE38KDEL was constructed successfully.The main product was in inclusion bodies.ELISA assay showed that the refolding recombinant immunotoxin remained binding activity with SEA antigen.Conclusion A new recombinant expression plasmid pBAD/gIII A-scfv-PE38KDEL has been constructed and expressed successfully,which is useful in further study of the treatment of schistosomiasis japonica.

关 键 词：日本血吸虫 SCFV PE38KDEL 重组免疫毒素 

分 类 号：R383.24[医药卫生—医学寄生虫学]