Anti HER2 ScFv-GFP融合抗体靶向检测HER2的表达  

Targeted detecting HER2 expression with recombinant anti HER2 ScFv-GFP fusion antibody

作　　者：高国辉[1] 陈翀[2] 杨艳梅[1] 杨涵[2] 王金丹[1] 郑易[1] 黄奇迪[2] 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,浙江温州325035 [2]温州医学院附属第一医院肿瘤外科,浙江温州325035

出　　处：《生物工程学报》2012年第8期1002-1014,共13页Chinese Journal of Biotechnology

基　　金：国家自然科学基金(No.30801118/C160403);浙江省自然科学基金(No.Y2110623);浙江省卫生厅医药卫生计划项目(No.2012KYA128)资助~~

摘　　要：为验证真核表达的携带绿色荧光的抗HER2单链抗体应用于临床诊断HER2阳性肿瘤细胞和病理组织的可靠性,构建融合基因Anti HER2 ScFv-GFP,重组入pFAST Bac HT A载体,在昆虫细胞Sf9中表达,以Ni2+-NTA亲和层析法纯化Anti HER2 ScFv-GFP融合蛋白,测定其浓度与纯度,将同浓度的纯化蛋白分别与3种乳腺癌细胞BT474、SKBR3和MCF7各混合12 h、24 h和48 h,分析其在不同时间段结合HER2阳性肿瘤细胞的稳定性。用纯化蛋白直接检测经抗原修复的乳腺癌病理组织,与免疫组织化学法检测结果对比。结果在昆虫细胞Sf9中可观察到明显绿色荧光,纯化的融合蛋白相对分子量约60 kDa,浓度为115.5μg/mL,纯度约97%,SKBR3和BT474鉴定为HER2阳性。结合12 h、24 h、48 h后其细胞表面均有明显绿色荧光,而HER2阴性MCF7被洗脱后无荧光,该抗体滴度为1:64,48 h内该荧光抗体仍具有稳定性。携带绿色荧光的融合抗体检测病理组织与IHC法的结果完全一致。表明成功表达的携带绿色荧光的抗HER2单链抗体可特异性检测HER2阳性乳腺癌细胞BT474和SKBR3,在HER2阳性肿瘤细胞和临床病理组织检测上具有应用前景。To verify the reliability of targeted detecting HER2 positive cancer cells and clinical pathological tissue specimens with a recombinant anti HER2 single chain antibody in single chain Fv fragment（scFv） format,we have constructed the fusion variable regions of the ScFv specific for HER2/neu.labeled a green-fluorescent protein（GFP）.The humanized recombinant Anti HER2 ScFv-GFP gene was inserted into pFast Bac HT A,and expressed in insect cells sf9.Then the recombinant fusion protein Anti HER2 ScFv-GFP was properly purified with Ni2＋-NTA affinity chromatography from the infected sf9 cells used to test the specificity of the fusion antibody for HER2 positive cancer cells.Firstly,the purified antibody incubated with HER2 positive breast cancer cells SKBR3,BT474 and HER2 negative breast cancer cells MCF7 for 12 h/24 h/48 h at 37 °C,in order to confirm targeted detecting HER2 positive breast cancer cells by Laser Confocal Microscopy.Furthermore,the same clinical pathological tissue samples were assessed by immunohistochemistry（IHC） and the fusion antibody Anti HER2 ScFv-GFP in the meanwhile.The data obtained indicated that the recombinant eukaryotic expression plasmid pFast Bac HT A/Anti HER2 ScFv-GFP was constructed successfully In addition,obvious green fluorescent was observed in insect cells sf9.When the purified fusion antibody was incubated with different cancer cells,much more green fluorescent was observed on the surface of the HER2 positive cancer cells SKBR3 and BT474.In contrast,no green fluorescent on the surface of the HER2 negative cancer cells MCF7 was detected.The concentration of the purified fusion antibody was 115.5 μg/mL,of which protein relative molecular weight was 60 kDa.The analysis showed the purity was about 97% and the titer was about 1：64.The detection results of IHC and fusion antibody testing indicated the conformity.In summary,the study showed that the new fusion antibody Anti HER2 ScFv-GFP can test HER2 positive cancer cells,indicating a potential candidate method for cl

关 键 词：抗HER2单链抗体 绿色荧光蛋白 融合抗体 HER2阳性肿瘤细胞 HER2检测功能 分子诊断 

分 类 号：R730.4[医药卫生—肿瘤]