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目的利用分泌具有GPX活性的单克隆抗体(mAb)的杂交瘤细胞3G5,克隆其mAb体的可变区基因。方法提取杂交瘤细胞的总RNA ,分离mRNA ,反转录合成cDNA。经PCR扩增VH 基因和VL 基因 ,将VH 基因和VL基因与载体 pGEM T连接后 ,进行酶切鉴定和序列分析。结果构建了2个分别含有VH 和VL 基因的重组质粒。序列分析表明 ,VH 和VL 分别属于mouscheavychainsubgroupIII和mouselightchainsubgroupV亚群 ,长度为372和324bp ,编码124和108个氨基酸 ,在高变区分别有4和2个丝氨酸。结论克隆的VH 和VL 基因符合功能性重排的鼠抗体可变区基因特征 ,为将来制备具有GPX活性的单链抗体提供了可靠的基因材料。
Objective To clone the variable region genes of their mAb bodies using hybridoma cells 3G5 secreting monoclonal antibodies (mAbs) having GPX activity. Methods Total RNA was extracted from hybridoma cells, mRNA was isolated and cDNA was reverse transcribed. The VH gene and the VL gene were amplified by PCR, and the VH gene and the VL gene were ligated with the vector pGEM T to carry out restriction analysis and sequence analysis. Results Two recombinant plasmids containing the VH and VL genes were constructed. Sequence analysis showed that VH and VL belong to the subtypes mouscheavychainsubgroupIII and mouselightchainsubgroupV, respectively, with lengths of 372 and 324 bp, encoding 124 and 108 amino acids and 4 and 2 serines in hypervariable regions respectively. Conclusion The cloned VH and VL genes are consistent with the functional rearrangement of murine antibody variable region genes and provide reliable genetic material for the future preparation of single chain antibodies with GPX activity.