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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 prokaryotic fusion expression vector of the monoclonal antibody against NP11-4 against Schistosoma japonicum and the PE38KDEL from Pseudomonas exotoxin, and to identify its biological activity. Methods The NP11-4VH, VL, scFv and PE38KDEL genes were amplified by PCR in vitro. After sequencing, the scFv and PE38KDEL genes were digested with restriction endonucleases and ligated to construct the prokaryotic expression vector pBAD / gIIIA-scFv-PE38KDEL , Transformed into E. coliTop10F ’, L-arabinose induced expression, the expression product was identified by Western blot and ELISA activity. Results Recombinant immunotoxin expression vector pBAD / gIIIA-scFv-PE38KDEL was constructed. The expressed product was mainly in the form of inclusion bodies with a molecular weight of about 70 kD. The recombinant immunotoxin after the denaturation and refolding was better than that of the soluble antigen Of binding activity. Conclusion The construction and expression of the immunotoxin against S. japonicum single-chain antibody provides a new idea for the treatment of schistosomiasis.