Anti-HER2-ScFv-GFP融合蛋白靶向结合体外乳腺癌细胞表面受体的研究  

The Study of the Targeting Selectivity and Binding the Surface of Breast Cancer Cells with the Fusion Protein Anti-HER2-ScFv-GFP in vitro Experiments

作　　者：高国辉[1] 黄奇迪[2] 王金丹[1] 杨纪锋[1] 包兵兵[1] 胡孝渠[2] 

机构地区：[1]温州医学院生命科学院,浙江省医学遗传学重点实验室,温州325035 [2]温州医学院附属第一医院肿瘤外科,温州325035

出　　处：《中国细胞生物学学报》2011年第5期485-491,共7页Chinese Journal of Cell Biology

基　　金：国家自然科学基金(No.30801118/C160403);浙江省自然科学基金委(No.Y207301);温州市科技局(No.Y20090293;No.Y2003A138)资助项目~~

摘　　要：为了研究不同表达系统获得的携带绿色荧光抗HER2单链抗体(Anti-HER2-ScFv-GFP)是否既可靶向结合HER2阳性乳腺癌细胞表面,也可通过观察绿色荧光变化直接判断抗体结合乳腺癌细胞表面后细胞的动态变化,在前期成功构建两种表达系统的基础上,利用Ni^(2+)-NTA亲和层析法纯化来源于真核表达系统pFAST Bac to Bac HT A/Tn-5B1-4和原核表达系统pBAD His B/TOP10的融合蛋白Anti-HER2-ScFv-GFP,设置HER2阳性细胞SKBR3为实验组、HER2阴性细胞MCF7为对照组,分别与之混合24 h后,1×PBS洗脱细胞3次,激光共聚焦显微镜观察到两种不同表达系统获得的融合蛋白在HER2阳性细胞SKBR3表面分布均有绿色荧光,真核表达的蛋白结合效率明显高于原核表达的蛋白,SKBR3结合高浓度的融合蛋白后细胞表现出皱缩,绿色荧光明显增强,而两种不同来源的融合蛋白与HER2阴性MCF7混合后均易被洗脱。GFP标准品与SKBR3混合后也容易被洗脱。实验表明构建的携带绿色荧光抗HER2单链抗体同时具有靶向结合和报告作用两方面的功能。The goal of this study was to test the targeting binding efficiency of the fusion protein Anti- HER2-ScFv-GFP on the surface of breast cancer cells. We constructed the eukaryotic expression system pFAST Bac to Bac HT A/Anti-HER2-ScFv-GFP/Tn-5B1-4 and the prokaryotic system pBAD His B/Anti-HER2-ScFv- GFP/TOP10. And then the fusion protein Anti-HER2-ScFv-GFP was separated to get the purification with Ni〉- NTA argrose from the eukaryotic expression system pFAST Bac to Bac HT A/Tn-5B1-4 and the prokaryotic expres- sion system pBAD His B/TOP10. Then we incubated SKBR3 （HER2＋ cell） and MCF7（HER2-cell） containing the purification of the fusion proteins in 24 h, eluted these cells with 1 xPBS three times, examined the targeting bind- ing efficiency of the fusion protein Anti-HER2-ScFv-GFP on the surface of breast cancer cells with laser confocal microscopy system. Consequently, apparent green fluorescence was detected in SKBR3 cells. Fusion proteins from eukaryotic expression system showed a higher binding efficiency than those from prokaryotic expression system. Incubation with high concentration fusion proteins induced shrinking in SKBR3 cell. In contrast, fusion proteins were readily eluted from the HER2 negative cell MCF7, without obvious fluorescence detected. The standard GFP was readily eluted from the HER2 positive cell SKBR3, too. Fusion protein （Anti-HER2-ScFv-GFP） from these two systems can all bind to the surface of SKBR3 cell, but proteins from eukaryotic system showed a higher binding capacity than those from prokaryotic system. This suggested that GFP can report the developing of the breast cancer cells SKBR3 with anti HER2 ScFv and engineer antibodies selected to co-target critical functional pairs of HER2 on the surface of SKBR3 in vitro.

关 键 词：融合蛋Anti—HER2—ScFv—GFP 乳腺癌细胞表面受体 靶向结合 

分 类 号：R737.9[医药卫生—肿瘤]