日本血吸虫单克隆抗抗独特型抗体NP48单特异性双链抗体的构建表达与初步鉴定

目的构建和表达日本血吸虫单克隆抗抗独特型抗体NP48单特异性双链抗体，并初步鉴定表达产物活性。方法应用SOE法扩增双链抗体基因VH-linker—VL（Diabody，简称D）。将D重组入原核表达载体pBAD／g Ⅲ。表达质粒转化E、coli TOP10，左旋阿拉伯糖诱导表达。对D的表达产物进行分离纯化，采用Dot—ELISA法检测纯化蛋白与NP30、可溶性虫卵抗原（SEA）、可溶性成虫抗原（SWAP）的结合活性。结果测序证实双链抗体基因D构建成功；构建了D的原核表达系统，诱导表达产物主要以包涵体形式存在，分子量约为27kD；经纯化、变性复性后用Dot—ELISA法鉴定结果表明，D表达的纯化蛋白可与NP30特异性结合。结论所构建、表达的NP48单特异性双链抗体具有亲本单抗的部分特性。

Construction, expression and characterization of a mono-specific bivalent diabody derived from an anti-anti-idiotypic monoclonal antibody NP48 of Schistosoma japonicum

Objective To construct a mono-specific bivalent diabody (scFv dimer) gene derived from an anti-anti-idiotypic monoclonal antibody NP48 of Schistosoma japonicum and to express and characterize the protein.MethodsThe mono-specific diabody gene (D) was constructed by SOE (splicing by overlap extension) and using Gly_4Ser as a linker to join the C-terminus of the V_H to the N-terminus of the V_L.D was linked with prokaryotic expression vector pBAD/g. The target protein expression in E.coli TOP10 was induced by arabinose. Then a purification procedure for the target protein was carried out. The antigen binding activity of expressed product was detected with Dot-ELISA. ResultsThe V_H-G_4S-V_L (D) gene was confirmed by sequencing. The pBAD/g-D recombinant were determined by digesting with endonucleases and expected bands were identified. There were less soluble target proteins in the supernantes and higher target proteins in the pellets as inclusion body when separating the D expression proteins. And the insoluble fraction was recovered as a soluble, correctly processed protein by solubilising with 8 mol/L Urea. The molecular weight of the target protein was about 27 kD. The binding activity of the target protein to anti-idiotypic monoclonal antibody NP30 of Schistosoma japonicum was verified by Dot-ELISA. ConclusionThe purified protein from the constructed recombinant pBAD/g-D could interact specifically with antigen NP30. So the constructed mono-specific diabody has the part characteristics of anti-anti-idiotypic monoclonal antibody NP48 of Schistosoma japonicum.