融合蛋白anti-HER2-ScFv-GFP在昆虫细胞中的表达及其靶向结合乳腺癌细胞的功能分析  

Expression of fusion protein anti-HER2-ScFv-GFP in insect cells and binding to surface of breast cancer cells

作　　者：高国辉[1] 王金丹[1] 黄奇迪 杨纪峰[1] 杨水兵[1] 包兵兵 张羽 胡孝渠 

机构地区：[1]温州医学院生命科学院 [2]生命科学院附属第一医院肿瘤外科 [3]仁济学院,浙江温州325000

出　　处：《中国病理生理杂志》2011年第5期1034-1040,共7页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.30801118/C160403);浙江省自然科学基金资助项目(No.Y207301);温州市科技局资助项目((No.Y20090293No.Y2003A138)

摘　　要：目的:利用昆虫细胞-杆状病毒表达系统表达获得携带绿色荧光蛋白(GFP)的抗人表皮生长因子受体2(HER2)的单链抗体可变区片段(ScFv),分析其靶向结合乳腺癌细胞表面受体HER2的功能。方法:构建融合基因anti-HER2-ScFv-GFP,重组获得真核表达载体重组子pFAST Bac-to-Bac HT A/anti-HER2-ScFv-GFP,转化DH10Bac,收集重组病毒bacmid,分别转染、感染粉纹夜蛾细胞Tn-5B1-4,SDS-PAGE及Westernblotting分析融合蛋白表达产物。Ni2+-NTA亲和层析法纯化anti-HER2-ScFv-GFP融合蛋白后分别滴入乳腺癌细胞HER2阳性的SKBR3和HER2阴性的MCF7,对比携带绿色荧光的抗HER2单链抗体靶向结合乳腺癌细胞表面HER2受体情况。结果:获得长度约1 539 bp的融合基因anti-HER2-ScFv-GFP,感染重组病毒的Tn-5B1-4细胞膜附近有明显绿色荧光,SDS-PAGE、Western blotting法检测到60 kD的特异性条带。结合实验显示HER2阳性的SKBR3细胞表面有明显绿色荧光,而HER2阴性的MCF7细胞表面荧光易被洗脱。结论:在Tn-5B1-4中成功表达携带绿色荧光的融合蛋白anti-HER2-ScFv-GFP,该携带绿色荧光的抗HER2单链抗体具有靶向结合乳腺癌细胞表面HER2受体的效能。AIM： To express a recombinant fusion protein anti-human epidermal growth factor receptor-2 single-chain variable fragment with green fluorescent protein（anti-HER2-ScFv-GFP） using the insect cells-Bac-to-Bac baculovirus expression system and to analyze the binding function of this fusion protein with HER2 on the surface of the breast cancer cells.METHODS： Human anti-HER2-ScFv gene from mice was fused with GFP gene.To obtain the recombinant plasmid pFAST Bac-to-Bac HT A/anti-HER2-ScFv-GFP,we inserted it into Bac-to-Bac baculovirus expression plasmid pFAST Bac-to-Bac HT A.The identified recombinant plasmid was transferred into Escherichia coli DH10Bac to allow the generation of a recombinant bacmid.After transfected the recombinant virus bacmid into the insect cells Tn-5B1-4,the recombinant virus was collected to infect Tn-5B1-4.SDS-PAGE and Western blotting analysis were used to verify the expression product in Tn-5B1-4.The fusion protein was purified with Ni2＋-NTA affinity chromatography.The purified fusion protein was bound to the surface of HER2-positive breast cancer cells SKBR3 and HER2-negative breast cancer cells MCF7.The binding effects on the surface of breast cancer cells were observed under laser confocal microscope.RESULTS： The fusion gene anti-HER2-ScFv-GFP was successfully constructed with the length of 1 539 bp.The green fluorescence was also observed in Tn-5B1-4 cells infected with the recombinant virus under fluorescent microscope.A 60 kD protein was examined and confirmed by SDS-PAGE and Western blotting.Under laser confocal microscope,strong green fluorescence was observed on the surface of the HER2-positive breast cancer cells SKBR3.However,no green fluorescence was observed on the surface of HER2-negative breast cancer cell MCF7.Obvious green fluorescence on the surface of HER2-positive breast cancer cell SKBR3 was also found after the cells were eluted with 1×PBS.CONCLUSION： The fusion protein anti-HER2-ScFv-GFP was successfully expressed in insect cells Tn-5B1-4,and can firmly

关 键 词：抗HER2单链抗体可变区片段 绿色荧光蛋白 融合基因表达 

分 类 号：R363[医药卫生—病理学]