抗人血管内皮生长因子(VEGF)单克隆抗体轻链可变区基因的克隆和序列分析

Clone and Sequence Characterization of Variable Region of Light Chain from Hybridoma Cells Secreting MAB Against VEGF

为构建和表达抗人血管内皮生长因子（ＶＥＧＦ）工程抗体奠定基础，从分泌抗人ＶＥＧＦ抗体的杂交瘤细胞株中提取总ＲＮＡ，利用反转录－聚合酶链反应方法，克隆抗人ＶＥＧＦ单克隆抗体轻链可变区基因，将其重组入ｐＥＧＭＲ－ＴＶｅｃｔｏｒ测序载体测序。结果表明轻链可变区序列全长３３３ｂｐ，编码１１１个氨基酸，通过国际联机检索及Ｋａｂａｔ库扫描证实，该轻链可变区符合小鼠免疫球蛋白轻链可变区基因特征，同源性高达８９．３％，归属小鼠轻链可变区基因第Ⅲ亚组。

To clone the variable rigion of light chain (VL) gene from hybridoma secreting monoclonal antibody(MAB) against human vasoendothelial growth factor (VEGF) for construction of recombinant antibody(ScFv). One method was used to extract total RNA, degeneration primer was used to amplify the cDNAs by retropolymerase chain reaction (RT PCR). The products were inserted to T vector and its sequence was analysed. Results showed that the VL gene was 333bp encoding 111 amine acid resides, and analysis of the nucleotide sequence of the light chain V region revealed that the VL sequence was 89.3% identical to the sequence present in mouse Ig VL region. According to kabat classfication, the VL gene belongs to mouse kappa light chains subgroup Ⅲ, resulting from VL Jκ 2 rearrangment.