摘要
设计并合成多个 6 0bp左右的DNA小片段 ,经重叠延伸PCR扩增获得 16 4 0bp的抗CD3 抗CD2 0双特异性单链抗体完整基因片段 ,将其克隆入真核定点表达载体pcDNA5 FRT中 ,脂质体法转染Flp_InTM CHO细胞 ,获得稳定表达细胞株 ,目的蛋白在上清中的表达量约为 30 0 μg L。采用Ni_NTA柱对其进行了纯化 ,经SDS_PAGE蛋白电泳及Western_blot分析结果表明 ,含组氨酸标签的目的蛋白的分子量约为 70kD ,与预期结果一致。活细胞间接免疫荧光实验和玫瑰花环实验证明抗CD3 抗CD2 0双特异性单链抗体具有与Ramous(CD2 0 +)及Jurkat(CD3+)细胞特异性结合的活性。光学显微镜下可以观察到抗CD3 抗CD2 0双特异性单链抗体可以有效介导人外周血淋巴细胞裂解B淋巴瘤细胞Ramous。以上工作为进一步了解抗CD3 抗CD2 0双特异性单链抗体的体内体外生物学活性奠定了基础。
The synthetic gene with 1640bp encoding for the anti-CD3 /anti-CD20 bispecific single-chain antibody was designed and obtained by SOE (sp licing by overlap extension) PCR. The cDNA was cloned into Flp-In TM expres sion vector pcDNA5/FRT and transfeced into Flp-In TM CHO cells to generate a stable expression cell line with a capacity for expressing anti-CD3/anti-CD20 bispecific single-chain antibody at 300μg/L. The protein, which had a molecular weight of about 70 kD,was purified by Ni-NTA affinity chromatography and ident ified by SDS-PAGE and Western-blot analysis. Immunofluorescence assay and cellul ar rosetting showed that it can react specifically on Jurkat(CD3+) and Ramous(CD 20+) cells. The lysis of human PBL against CD20-positive lymphoma Ramous cells i n the presence of the anti-CD3/anti-CD20 bispecific single-chain antibody can ob served by microscope. All these results would lighten the further study of its b iological functions in vitro and in vivo.
出处
《生物工程学报》
CAS
CSCD
北大核心
2005年第2期289-293,共5页
Chinese Journal of Biotechnology
基金
国际科技合作重点项目计划资助 (No.2 0 0 1AA2 15 461)~~
关键词
双特异性单链抗体
表达
坚定
活性分析
bispecific single-chain antibody,expression,identi fication, bioactivity characterization
