摘要
通过重组PCR构建了抗HER2单链抗体基因、绿脓杆菌外毒素 (PE)转位肽序列和活性型粒酶B(GrBa)基因相融合的sFv2 3e PEⅡ GrBa基因 ,以及N端包含PE部分转位肽序列的PEⅡ GrBa基因 .将这 2种粒酶B嵌合蛋白基因瞬时转染或稳定转染HeLa细胞及SKBR 3细胞 .通过间接免疫荧光、细胞计数、MTT、ELISA等方法 ,观察到细胞浆中表达的PEⅡ GrBa蛋白直接杀伤其表达细胞 ;而sFv2 3e PEⅡ GrBa表达后被分泌至细胞外 ,对产生它的细胞没有杀伤性 ,但能够特异识别并杀伤HER2阳性肿瘤细胞 .结果表明 ,抗肿瘤表面抗原的抗体能够介导靶向识别 ,转位结构域可以辅助效应分子活化、转位至细胞液并杀伤细胞 ,为肿瘤的靶向治疗提供了新的策略 .
Two chimeric human granzyme B genes were constructed by the recombinant PCR. One consisted of the genes of a single-chain antibody against HER2, a translocating sequence of Pseudomonas exotoxin A (PE) and active granzyme B(GrBa), termed sFv23e-PE Ⅱ-GrBa.The other, PE Ⅱ-GrBa, encoded portion of PE translocating domain and active granzyme B. These genes were then transiently or stably transfected into HeLa cells or SKBR-3 cells,respectively. As shown in indirect immunofluorescence, cell counting, MTT and ELISA analyses, the expressed PE Ⅱ-GrBa proteins were localized in the cytoplasm and cytotoxic to the cells, whereas sFv23e-PE Ⅱ-GrBa proteins were produced and secreted into the culture media, and selectively killed HER2-positive tumor cells but not the cells that produced them. The results provide a strategy for targeted cancer therapy in which effectors can be directed to the surface of tumor cells by antibodies recognition with tumor-specific antigens, and the effectors may translocate to the cytosol to cause target cell death via membrane-disruptive activities of translocating domains.
出处
《中国生物化学与分子生物学报》
CAS
CSCD
北大核心
2004年第5期637-642,共6页
Chinese Journal of Biochemistry and Molecular Biology
基金
国家高技术"863"计划资助项目 (No.2 0 0 1AA2 1710 1)
国家杰出青年科学基金资助项目 (No .3 992 5 0 3 6)
全军医药卫生科研基金重点项目 (No .0 1Z0 90 )~~