日本血吸虫单克隆抗独特型抗体NP30单链抗体基因的构建和表达

目的　构建日本血吸虫单克隆抗独特型抗体NP30单链抗体 (scFv)基因。　方法　通过PCR方法体外扩增并经测序验证的重链、轻链可变区 (VH、VL)基因先后重组入原核表达质粒 pTHA90相应的位点上 ,中间通过一连接肽 (Gly4 Ser) 3基因连接构建成单链抗体基因 (scFv) ,连接产物转化相应受体菌Top1 0 ,提取质粒 ,酶切鉴定重组克隆。表达产物经ELISA方法测定活性。　结果　重组克隆经酶切鉴定可见预期大小的片段 ,表明重组成功。表达产物经ELISA检测 ,OD4 92 值为 1 .0 6 ,高于阴性对照 3倍以上 ,证实具有与相应抗原结合的能力。　结论　成功地构建了scFv基因 ,且其表达产物保留了抗体的亲和性和特异性

Construction and Expression of Single Chain Fv Gene of Anti- idiotypic Monoclonal Antibody NP30 of Schistosoma japonicum

Objective To construct single chain Fv (scFv) gene of anti idiotypic monoclonal antibody NP30 of Schistosoma japonicum . Methods The heavy and light chain variable region genes of anti idiotypic monoclonal antibody NP30 of Schistosoma japonicum were inserted into two corresponding sites of expression vector pTHA90,and a scFv gene was constructed with a short peptide (Gly 4Ser) 3 linker gene. The recombinants were determined by digesting with Xho I/ Spe I, Xba I/ Eco RI and Xho I/ Eco RI,and then were introduced into E.coli Top10. The antigen binding activity of expressed product was detected with ELISA. Results The recombinants were determined by digesting with endonucleases and expected bands were identified. The value of expressed scFv was 3 times higher than negative control by ELISA(OD 492 =1.06). Conclusion The scFv gene of anti idiotypic monoclonal antibody NP30 of Schistosoma japonicum was successfully cloned, and the expressed scFv fragment could interact specifically with antigen NP48.