抗人前列腺干细胞抗原单克隆抗体可变区基因的5'RACE扩增及序列分析

Cloning and Analysis of the Sequence of Variable Region of An ti-Human PSCA Monoclonal Antibodies by 5'RACE

目的:克隆并分析抗人前列腺干细胞抗原单克隆抗体轻链和重链的可变区基因。方法:从分泌抗人前列腺干细胞抗原单克隆抗体的杂交瘤细胞株中提取总RNA,根据小鼠IgG恒定区序列设计特异性引物,通过5′RACE法扩增其轻链和重链的可变区基因,克隆入pMD18-T载体,测序并分析其可变区序列。结果:3株抗人前列腺干细胞抗原单克隆抗体的重链可变区基因序列全长均为423 bp,编码141个氨基酸残基;轻链可变区基因序列全长均为393 bp,编码131个氨基酸残基;在GenBank中对氨基酸序列进行比对分析,均符合小鼠IgG可变区基因的特征;根据Kabat法则对3株抗体轻链和重链可变区氨基酸序列进行分析,确定了3个抗原互补决定区、4个框架区和前导肽。结论:通过5'RACE法得到了3株抗人前列腺干细胞抗原单克隆抗体轻链与重链可变区基因,为进一步研究抗体三维结构、人源化改造奠定了基础。

Objective： To clone and analyze the genes of light chain variable region（VL） and heavy chain vari-able region（VH） of monoclonal antibody（mAb） against human prostate stem cell antigen（PSCA）. Methods： Total RNA was extracted from hybridoma cells which secrete mAb against human PSCA. Then VH and VL genes were amplified by 5＇RACE with specific primers designed to match the high conservative nucleotide sequence of con-stant regions of mouse IgG. The PCR products were inserted into pMD18-T vector, followed by sequencing and analysis. Results： For each anti-PSCA mAb, VH gene contained 423 bp and encoded 141 amino acids, and the VL gene contained 393 bp and encoded 131 amino acids. They were homologous with the variable region of mouse IgG in GenBank. According to Kabat＇s rules, there were 4 framework regions, 3 complementarity determin-ing regions and a leader sequence in the VH and VL gene respectively. Conclusion： Acquisition of genes of the VH and VL of the anti-human PSCA mAbs will be a experimental basis for subsequent study of the mAb＇s 3D structure or humanization of antibody.