抗人CD16单克隆抗体可变区基因的克隆和表达

目的：克隆抗人CD16单克隆抗体重，轻链可变区（VH，VL）基因并合成单链抗体（ScFv)基因。方法：从分泌抗人CD16单克隆抗体的杂交瘤细胞B88－9中提取总RNA，应用RT－PCR技术获得抗CD16单克隆抗体的VH，VL基因，用连接肽（Linker)肽VH和VL连接成具有VH－Linker-VL结构的ScFv基因，将其克隆到表达载体pcDNA3.1( )，并转杂COS－7细胞。结果：VH基因长度为354bp，属于鼠抗体可变区重链基因家族I（B）亚群，VL基因长度为333bp，属于鼠抗体可变区kappa轻链基因家族Ⅲ亚群，采用夹心ELISA方法检测到ScFv的表达。结论：抗人CD16单克隆抗体VH与VL基因的克隆和ScFv基因的构建为基于CD16的导向免疫治疗奠定了基础。

Cloning and expression of the variable region genes of the monoclonal antibody against human CD16

ve To clone the variable region genes of the monoclonal antibody against human CD16 and assemble ScFv gene. Methods RT-PCR was used to clone the immunoglobulin (Ig) genes encoding variable regions of heavy and light chains (VH and VL ) from the hybridoma cell line B88-9, which secrets the monoclonal antibody against human GD16 . The gene splicing by overlap extension was used to assemble a gene encoding the anti-CD16 ScFv. The ScFv was cloned into expression vector pcDNA3. 1 ( ) and expressed in COS-7 cells . Results The VH gene consists of 354 bp and the deduced amino acid sequence was closely related to the published sequences of the variable region subgroup I(B) of mouse Ig heavy chain. The VL gene consists of 333 bp and the deduced amino acid sequence was very similar to the published sequences of the variable region subgroup III of mouse Ig kappa light chain. The ScFv was detected by sandwich ELISA. Conclu-sion This study would make substantial contribution to the potential development of antibody-directed immunotherapy based on CD16.