抗日本血吸虫单克隆抗体NP11-4人／鼠嵌合Fab抗体片段的制备及功能鉴定

Construction,expression and characterization of chimeric mouse-human Fab of monoclonal antibody NP11-4 against Schistosomiasis japonica

目的:构建日本血吸虫单克隆抗体NP11-4的人/鼠嵌合Fab(cFab)抗体片段,并对cFab抗体片段结合活性进行鉴定。方法:用抗体框架区的通用引物PCR扩增抗日本血吸虫单克隆抗体NP11-4的VH及VL基因,测序分析其核苷酸序列。将测序正确的鼠源VH、VL分别与人IgG1的CH1、CL通过Overlap PCR扩增Fd及全长L,再利用Overlap PCR以Fd和全长L基因为模板扩增cFab基因。将cFab基因片段克隆至载体pComb3XSS中构建表达载体pComb3XSS-Fab,经异丙基-β-D-硫代半乳糖苷(IPTG)诱导进行蛋白可溶性表达。通过Westernblot和ELISA方法对cFab抗体片段进行检测。结果:构建出抗日本血吸虫单克隆抗体NP11-4 cFab表达载体,Westernblot结果证实在大肠杆菌Top10F′中表达出cFab可溶性抗体片段,ELISA结果显示cFab抗体片段与可溶性虫卵抗原(SEA)有较好的结合活性。结论:表达、纯化了抗日本血吸虫单克隆抗体NP11-4cFab抗体片段,cFab抗体片段具有与亲本抗体相同的抗原结合能力,为制备抗日本血吸虫人源化免疫毒素提供了技术贮备。

Objective：To construct chimeric mouse-human Fab antibody from a mouse monoclonal antibody NP11-4 which is specific to membrane protein of Schistosoma japonicum by genetic engineering technique,and further identify the binding ability of chimeric antibody fragment. Methods：Light chain variable region（VL） and heavy chain variable region（VH） genes cloned from mouse monoclonal antibody NP11-4 were amplified by RT-PCR, and sequenced and analyzed after being ligated into pMD18-T vector. Then V. and VL were fused into CH1 domain of human IgG1 and CL domain of human kappa chain respectively to obtain fragments of Fd and light chain. Chimeric Fab （cFab） was amplified by overlap PCR from chimeric genes of Fd and light chain. After the cFab DNA was ligated into the phagmid vector pComb3XSS,the expression vector pComb3XSS-Fab was transformed into E. coli Top 10F＇ ,and the of soluble protein induced by IPTG （isopropyl-β-D-thiogalactoside） was detected. The expressions of products were detected by ELISA and Western blot methods. Results：An expression vector pComb3XSS-Fab to express cFab against Schistosomiasis japonica was Successfully constructed. The cFab protein was expressed in soluble form and secreted form,which was confirmed by Western blot. ELISA demonstrated that the cFab possessed the activity and specificity of interacting with SEA（soluble egg antigen）. Conclusion：The cFab retained the high affinity and specificity similar with the original mouse mAb NP11-4. This cFab fragment can be further modified to generate immunotoxin for therapeutic uses.