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目的克隆抗人ＴＮＦ－α鼠单抗得可变区基因以构建人－鼠嵌合抗体表达载体。方法采用ＲＴ－ＰＣＲ技术，以前导肽序列的引物从１个分泌抗人ＴＮＦ－α的鼠单抗杂交瘤细胞系中克隆抗体轻链、重链可变区基因（Ｖκ，ＶＨ），在大肠杆菌中表达Ｆａｂ段核实其功能活性。结果分别得到了２个Ｖκ和２个ＶＨ基因。ＤＮＡ序列测定表明，其中１个轻链可变区基因为骨髓瘤细胞系中固有的无功能基因。１个重链可变区基因经原核系统表达测活表明无抗体活性。另一个轻链和重链可变区基因的成熟蛋白编码部分与从第一骨架区引物所克隆的、可在大肠杆菌表达出抗体活性的Ｖκ、ＶＨ序列相符。将该轻链、重链基因分别克隆到了人－鼠嵌合轻链、重链表达载体中。结论通过原核表达系统核实，获得了抗ＴＮＦ－α单抗的可变区基因
Objective To clone the variable region gene of anti-human TNF-α mouse monoclonal antibody to construct human-mouse chimeric antibody expression vector. METHODS: The light chain and heavy chain variable region genes (Vκ, VH) were cloned from a murine monoclonal antibody (McAb) secreting anti-human TNF-α using the primers of the leader peptide sequence by RT-PCR. The Fab fragment is expressed in bacilli to verify its functional activity. As a result, two Vκ and two VH genes were obtained respectively. DNA sequence analysis showed that one of the light chain variable region genes was an inherent non-functional gene in myeloma cell lines. The expression of a heavy chain variable region gene by prokaryotic expression assay showed no antibody activity. The mature protein-coding portion of the other light and heavy chain variable region genes is consistent with the VK, VH sequences cloned from the first framework region primers that express antibody activity in E. coli. The light and heavy chain genes were cloned into human-mouse chimeric light and heavy chain expression vectors, respectively. Conclusion The variable region gene of anti-TNF-α mAb was obtained through prokaryotic expression system verification