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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 human / mouse chimeric Fab (cFab) antibody fragment of Schistosoma japonicum monoclonal antibody NP11-4 and to identify the binding activity of cFab antibody fragment. Methods: The VH and VL genes of monoclonal antibody NP11-4 against Schistosoma japonicum were amplified by PCR using the universal primer of antibody framework region, and the nucleotide sequence was analyzed by sequencing. The correct sequencing murine VH, VL and human IgG1 CH1, CL by Overlap PCR amplification of Fd and full length L, and then use Overlap PCR Fd and full-length L gene as a template for cFab gene amplification. The cFab gene fragment was cloned into the vector pComb3XSS to construct the expression vector pComb3XSS-Fab. The soluble protein was induced by isopropyl-β-D-thiogalactoside (IPTG). The cFab antibody fragment was detected by Western blot and ELISA. Results: The expression vector of NP11-4 cFab against Schistosoma japonicum was constructed. The result of Western blot confirmed that cFab soluble fragment was expressed in E. coli Top10F ’. The results of ELISA showed that the fragment of cFab was highly soluble in soluble egg antigen (SEA) Of binding activity. CONCLUSION: The NP11-4cFab fragment of monoclonal antibody against Schistosoma japonicum is expressed and purified. The cFab antibody fragment has the same antigen binding ability as the parental antibody, which provides a technical reserve for the preparation of anti-Schistosoma japonicum humanized immunotoxin.