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目的 获取抗CD4 单克隆抗体 (McAb)可变区基因 ,为构建抗CD4 嵌合抗体打下基础。方法 从分泌抗人CD4 McAb的杂交瘤细胞系中提取总RNA ,用家族特异性引物进行逆转录多聚酶链反应 (RT -PCR) ,分别扩增出重链可变区 (VH)基因及轻链可变区 (VL)基因。将PCR产物克隆至pGEM -T载体 ,然后转化JM10 9细菌。并用全自动DNA序列分析仪对VH及VL基因序列进行测定。结果 VH基因为 3 5 1bp ,编码 117aa残基 ;VL基因为 3 3 3bp ,编码 111aa残基。根据Kabat分类体系 ,VH隶属小鼠免疫球蛋白 (Ig)重链Ⅱ (A)亚组 ,VL隶属小鼠Igκ链Ⅲ亚组。结论 从推导出的氨基酸序列证实 ,所获取的VH和VL序列均符合小鼠Ig可变区的氨基酸序列特征。
Objective To obtain the variable region genes of anti-CD4 monoclonal antibody (McAb) and lay the foundation for the construction of anti-CD4 chimeric antibody. Methods Total RNA was extracted from the hybridoma cell line secreting anti-human CD4 McAb. Family-specific primers were used for reverse transcriptase-polymerase chain reaction (RT-PCR) to amplify the variable heavy chain (VH) gene and light chain Variable region (VL) gene. The PCR product was cloned into the pGEM-T vector and then transformed into JM109 bacteria. The sequences of VH and VL genes were determined by automatic DNA sequence analyzer. The results showed that the VH gene was 35bp and encoded a 117aa residue. The VL gene was 33bp and encoded a 111aa residue. According to the Kabat classification system, VH belongs to the subgroup of mouse immunoglobulin (Ig) heavy chain Ⅱ (A), and VL belongs to the mouse Ig κ chain Ⅲ subgroup. Conclusion The deduced amino acid sequence confirmed that the obtained VH and VL sequences all fit the amino acid sequence of mouse Ig variable region.