摘要
目的 获得抗人CD25分子单抗的轻链及重链可变区基因,并进行序列分析。方法 采用RT- PCR,从WuTac细胞总RNA中扩增出轻链、重链可变区基因,进行克隆并作核苷酸序列分析。结果 构建的PMD18 -T-VL和PMD18-T-VH重组克隆载体,酶切与预期结果相符。VH和VL基因序列与鼠抗体的同源性大于 80%。克隆的重链可变区基因长度为351bp,属于鼠重链Ⅲ(C)亚类。轻链可变区基因长度为324bp,属于鼠轻链 Ⅳ 亚类。结论 本研究采用RT-PCT法扩增出轻链及重链可变区基因,并进行了序列测定和分析,为进一步构建嵌 合抗体以及单链抗体奠定了基础。
Objective To obtain the VH and VL genes of CD25 McAb and analyze the sequences of them. Methods The VL and VH genes were amplified by RT - PCR from the total RNA of WuTac cell, then cloned into vector PMD18-T and transformed to E. coli JM109 The recombinant plasmids were identified by enzyme digestion , and positive clones were sequenced. Results The result of enzyme digestion was m consis-tent with expected. The cloned VL and VH genes, with the lengths of 324 and 351bp respectively, were belong to the subgroup Ⅳ of light chain and subgroup Ⅲ(C) of heavy chain of murine McAb respectively, and the homology of them to the variable region genes of murine CD25 McAb were above 80% . Conclsion The VH and VL genes of CD25 McAb were directly amplified by RT- PCR in this paper. The study laid a foundation of further development of chimeric and single chain antibodies.
出处
《中国生物制品学杂志》
CAS
CSCD
2001年第3期129-131,共3页
Chinese Journal of Biologicals