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目的进行ＴＮＦ－α单抗Ｆａｂ段基因的克隆并在大肠杆菌中表达。方法用逆转录－聚合酶链反应技术（ＲＴ－ＰＣＲ）从分泌抗人ＴＮＦ－α的鼠单抗杂交瘤细胞系中克隆重链Ｆｄ段和κ链基因；并用ＥＬＩＳＡ和免疫印迹分析证明表达的Ｆａｂ段特异结合ＴＮＦ－α。结果从分泌抗人ＴＮＦ－α的鼠单抗杂交瘤细胞系中克隆出了重链Ｆｄ段和κ基因。经ＤＮＡ序列测定表明，ＶＨ、Ｄ、ＪＨ分别属于ＶＨ３Ｄ、ＤＳＰ２和ＪＨ４；Ｖκ和Ｊκ分别属于Ｖκ１和Ｊκ１。将该Ｆｄ和κ链ｃＤＮＡ克隆到表达载体ｐＣｏｍｂ３Ｈ中，在大肠杆菌中获得了表达。结论ＥＬＩＳＡ和免疫印迹分析表明，表达的Ｆａｂ段可特异地和ＴＮＦ－α结合。
Objective To clone the Fab fragment of TNF-α monoclonal antibody and express it in Escherichia coli. Methods The heavy chain Fd fragment and κ chain gene were cloned from murine monoclonal antibody secreting anti-human TNF-α by reverse transcription-polymerase chain reaction (RT-PCR). The expression of Fab fragments specifically bind TNF-α. Results The heavy chain Fd segment and kappa gene were cloned from murine monoclonal antibody secreting anti-human TNF-α. DNA sequencing showed that VH, D and JH belonged to VH3D, DSP2 and JH4, respectively; Vκ and Jκ belonged to Vκ1 and Jκ1 respectively. The Fd and kappa chain cDNAs were cloned into the expression vector pComb3H and expressed in E. coli. Conclusions ELISA and immunoblot analysis showed that the expressed Fab fragments specifically bind to TNF-α.