Anti-HER2 ScFv-GFP融合蛋白在大肠杆菌中的表达及其靶向结合乳腺癌细胞的初步分析

Expression of the fusion protein anti-HER2 ScFv-GFP in E.coli and the analysis of targeting binding function on the surface of the breast cancer cells

目的:构建原核表达系统诱导获得携带绿色荧光的抗人表皮生长因子受体2(human epidermalgrowth factor receptor 2,HER2)单链抗体,分析其靶向结合HER2阳性肿瘤细胞的功能,探讨其应用于HER2阳性肿瘤分子诊断与靶向治疗的可能性。方法:将绿色荧光蛋白基因与鼠源性人抗HER2单链抗体基因拼接成融合基因anti-HER2 ScFv-GFP,构建原核表达载体重组子pBAD His B/anti-HER2-ScFv-GFP,转入大肠杆菌E.coli TOP10诱导表达,SDS-PAGE和Western Blot法鉴定,Ni2+-NTA亲和层析法纯化获得目的融合蛋白。将不同浓度的融合蛋白anti-HER2 ScFv-GFP分别与HER2阳性的乳腺癌细胞SKBR3、HER2阴性的MCF7混合,观察绿色荧光在不同癌细胞表面的分布。结果:成功获得长度为1 539 bp的融合基因,原核表达系统pBAD His B/anti-HER2-ScFv-GFP/TOP10成功构建并表达携带绿色荧光的抗HER2单链抗体,融合蛋白相对分子量大小约60 kDa。HER2阳性的SKBR3细胞表面有明显绿色荧光,细胞有皱缩现象,而HER2阴性MCF7被洗脱后无荧光。结论:携带绿色荧光的抗HER2单链抗体能牢固结合在HER2阳性的乳腺癌细胞SKBR3表面,绿色荧光可以起到报告作用。

Objective： To construct the prokaryotic expression system pBAD His B/anti-HER2-ScFv-GFP/TOP10 and get the fusion protein anti-human epidermal growth factor receptor-2 single chain Fv antibody with green fluorescent protein,and then analyze the probability of fusion protein anti-HER2 ScFv-GFP＇s targeting binding function on the HER2 positive breast cells.Methods： Human anti-HER2 single chain antibody gene fragments from mice was fused with green fluorescent protein gene.then expression recombinant plasmid vectors pBAD His B/anti-HER2 ScFv-GFP was constructed.The transgenic E.coli TOP10 were obtained that expressed humanized anti-HER2 ScFv induced by adding 0.2% L-Arabinose.SDS-PAGE was run with the recom-binant E.coli TOP10 culture supernatant and then transferred to nitrocellulose membrane for western blotting analysis.The recombinant fusion protein anti-HER2-ScFv-GFP was purified from the induced bacterial cells using Ni2＋-NTA affinity chromatography.The fusion protein with different concentration was mixed with the surface of HER2 positive breast cancer cells SKBR3 and HER2 negative breast cancer cells MCF7.The distribution on the surface of the different breast cancer cells was observed under the Laser Confocal Microscopy.Results： The fusion gene anti-HER2-ScFv-GFP was fused successfully.The length of the fusion gene was 1 539 bp.The prokaryotic expression system pBAD His B/anti-HER2-ScFv-GFP/TOP10 was successfully constructed.SDS-PAGE and western blotting analysis showed that the fusion protein was expressed.It indicated that the fusion protein anti-HER2-ScFv-GFP was 60kDa.The recombinant protein could be combined on the surface of HER2 positive breast cancer cell SKBR3 and green fluorescent appeared.But the fusion protein with green fluorescent was eluted using 1×PBS easily from HER2 negative breast cancer cell MCF7.Conclusion： the fusion protein has the binding function of targeting tumor.And the targeting binding efficency can be predict according to the green fluorescent.