日本血吸虫单克隆抗独特型抗体NP30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独特型抗体NP30单特异性双链抗体，初步鉴定表达产物的活性。方法用overlap PCR法扩增双链抗体基因VH-GGGGS—VL将双链抗体基因重组入原核表达载体pBAD／gⅢ。表达质粒转化E．coli TOP10F’，左旋阿拉伯糖诱导表达。对表达产物进行分离纯化，ELISA检测纯化蛋白与血吸虫病人血清抗体的结合活性。结果测序证实双链抗体基因正确，构建了双链抗体的原核表达系统，双链抗体在细菌超声上清和沉淀内均有表达，分子量约为34kD。纯化产物经ELISA鉴定，结果表明NP30单特异性双链抗体可与血吸虫病人血清抗体特异性结合。结论构建和表达的日本血吸虫单克隆抗独特型抗体NP30单特异性双链抗体具有与亲本单抗相同的结合活性。

Construction, expression and preliminary characterization of a mono-specific diabody derived from monocional anti-idiotypic antibody NP30 of Schistosoma japonicum

Objective To construct and express a mono-specific diabody derived from a monoclonal anti-idiotypic antibody NP30 of Schistosoma japonicum and characterize the protein. Methods The mono-specific diabody gene was constructed by overlap PCR and using Gly4Ser as a linker to join the VH to the VL. The diabody gene was linked with prokar;yotic expression vector pBAD/gIII, and the recombinant was transformed to E. coli TOP10F＇. The target protein expression was induced by L-Arabinose. Then a purification procedure for the target protein was performed. The antigen binding activity of expressed protein was detected with ELISA. Results The mono-specific diabody gene was confirmed by sequencing. There were less soluble target proteins in the supernates and higher target proteins in the pellets as inclusion body when separating the expression proteins. The molecular weight of target protein was about 34 kD. The binding activity of purificated protein with the blood serum of patients of Schistosoma japonicum was verified by ELISA. Conclusion The constructed mono-specific diabody has a similar affinity with monoclonal antiidiotypic antibody NP30 of Schistosoma japonicum.