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目的 将单链抗体基因插入单嗜性逆转录病毒胞膜蛋白 ,以构建能靶向感染肿瘤细胞的逆转录病毒载体。方法 构建单链抗体融合表达质粒PET2 0b scFv ,通过酶联免疫分析 ,Westernblot检测基因工程单链抗体的抗原结合活性。利用PCR定点突变技术 ,在野生型MoMulv胞膜蛋白上的SU亚单位氨基末端产生合适的酶切位点 ,插入体外构建 ,证实具有抗原亲合活性的抗人脑胶质瘤细胞膜抗原的单链抗体基因 ,并构建利用CMV启动子高效表达胞膜蛋白 单链抗体融合基因的表达质粒 ,转染Ψ2包装细胞后 ,以lac z为报告基因 ,包装出重组逆转录病毒 ,进行病毒结合实验和感染实验。结果 酶联免疫分析 ,Westernblot结果证实基因工程单链抗体能与肿瘤细胞表面相应抗原结合 ,病毒结合实验与感染实验证明所构建嵌合型逆转录病毒能改变其嗜性 ,由仅感染鼠细胞到特异性感染人脑胶质瘤细胞 ,且这种作用能为特异性单克隆抗体阻断。结论 利用单链抗体修饰单嗜性逆转录病毒胞膜蛋白的SU亚单位能达到选择性感染肿瘤细胞的目的。
Objective To insert a single-chain antibody gene into the cytotrophic protein of monotrophic retrovirus to construct a retroviral vector that can target infected tumor cells. Methods The single chain antibody fusion expression plasmid PET2b was constructed and its antigen binding activity was detected by enzyme-linked immunosorbent assay and Western blot. Using PCR site-directed mutagenesis, a suitable restriction enzyme site was generated at the amino terminus of the SU subunit on the wild-type MoMulv membrane protein and inserted into an in vitro construct to confirm that the anti-human glioma cell membrane antigen single chain Antibody gene was constructed and the expression plasmid for expressing the fusion protein of the single-chain antibody of cell membrane protein with CMV promoter was constructed. The transfected cells were transfected with Ψ2, and lacz was used as the reporter gene. The recombinant retrovirus was packaged and tested for virus binding and infection experiment. Results Enzyme-linked immunosorbent assay and Western blot showed that the single-chain antibody could bind to the corresponding antigen on the surface of tumor cells. The virus-binding and infection experiments demonstrated that the constructed chimeric retrovirus could change its tropism by infecting mouse cells only Specific infection of human glioma cells, and this effect can be blocked by specific monoclonal antibodies. Conclusion The single-chain antibody modified subunit of single tropism retroviral membrane protein can achieve the purpose of selective infection of tumor cells.