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本研究利用逆转录PCR技术和家族特异性引物,先后从9株分泌不同特异性单抗杂交瘤细胞株中扩增出了轻、重链可变区基因,并经克隆和序列测定。其中利用轻链引物(VL5.1和VL3.1)从分泌抗转铁蛋白受体(anti-transferrlin receptor)单抗的杂交瘤细胞株(7579)中扩增出了无功能的V_k序列。经分析和与GenBank数据库比较,该序列虽与BALB/cV_k21-E细胞株分泌的抗体轻链有98％同源性,但是在其V/J连接区内有4个碱基对缺失,导致后续阅读框架移位,因而出现终止密码子而致翻译停止。当改变引物(VL5.2和VL3(JK1/2))之后,又分别从该种杂交瘤细胞株中扩增出有功能的V_k基因序列。说明同种杂交瘤细胞中,部分细胞出现基因异常性重排(aberrant rearrangement),而这种重排的V-J基因片段来源于原融合细胞MOPC-21骨髓瘤细胞系。
In this study, the light and heavy chain variable region genes were amplified from nine hybridoma cell lines secreting different specific monoclonal antibodies using reverse transcription PCR and family-specific primers, and cloned and sequenced. Wherein a non-functional V_k sequence was amplified from a hybridoma cell line secreting an anti-transferrlin receptor monoclonal antibody (7579) using light chain primers (VL5.1 and VL3.1). After analysis and comparison with GenBank database, this sequence has 98% homology with the light chain of antibody secreted by BALB / cV_k21-E cell line, but has 4 base pair deletions in the V / J junction region, The reading frame is shifted so that the stop codon appears and the translation stops. After changing the primers (VL5.2 and VL3 (JK1 / 2)), the functional V_k gene sequences were amplified from the hybridoma cell lines respectively. This indicates that some cells in the same kind of hybridoma cells have aberrant rearrangement, whereas the rearranged V-J gene fragment is derived from the original fusion cell MOPC-21 myeloma cell line.