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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。
In order to verify the eukaryotic expression of green fluorescent anti-HER2 single chain antibody for the clinical diagnosis of HER2 positive tumor cells and pathological tissue reliability, the fusion gene Anti HER2 ScFv-GFP was constructed and recombined into pFAST Bac HT A vector, Sf9. Anti HER2 ScFv-GFP fusion protein was purified by Ni2 + -NTA affinity chromatography. The concentration and purity of Anti HER2 ScFv-GFP fusion protein were determined. The purified proteins were respectively mixed with three kinds of breast cancer cells BT474, SKBR3 and MCF7 for 12 h , 24 h and 48 h, the stability of HER2-positive tumor cells was analyzed at different time points. The purified protein was used to directly detect the pathological tissue of the breast cancer after antigen retrieval, and compared with the results of immunohistochemistry. Results The green fluorescent protein was observed in insect cells Sf9. The purified fusion protein had a relative molecular mass of about 60 kDa, a concentration of 115.5 μg / mL and a purity of about 97%. SKBR3 and BT474 were identified as HER2 positive. After 12 h, 24 h and 48 h, the cell surface was clearly green fluorescence, while HER2-negative MCF7 was eluted without fluorescence, the antibody titer was 1: 64,48 h the fluorescent antibody still has stability. The result of IHC assay that the green fluorescent fusion antibody detected the pathological tissue was completely consistent. The results showed that the green fluorescent anti-HER2 single chain antibody carrying EGFP could specifically detect HER2 positive breast cancer cells BT474 and SKBR3, and could be used for the detection of HER2 positive tumor cells and clinicopathological tissues.