CD25抗体轻链及重链可变区基因的克隆和序列分析

Cloning and Sequencing of V_H and V_L Genes of CD25 McAb

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.