抗恶性黑素瘤单链抗体-鱼精蛋白融合蛋白表达载体的构建及评评价

发布时间：2018-06-09 05:14

本文选题：恶性黑色素瘤 + 单链抗体　；参考：《南方医科大学学报》2017年09期

【摘要】：目的构建抗MM单链抗体-鱼精蛋白融合蛋白表达载体,并验证其表达效率及功能。方法通过PCR方法将鱼精蛋白截短片段序列与抗黑色素瘤细胞表面抗原单链抗体基因相连接;采用TaKaRap MD19-T载体试剂盒将PCR后获得的融合蛋白扩增产物进行T载体构建;构建GST融合蛋白表达载体以实现在大肠杆菌中该融合蛋白的可溶性表达,经两步亲和纯化后获得scFv-tP蛋白;采用凝胶阻滞电泳迁移率变动分析EMSA技术检测Anti-MM scFv-tP与siRNA的结合活性;荧光分子标记scFv-tP蛋白后分别采用流式细胞术检测和共聚焦显微镜观察其与Li Br细胞株进行细胞表面抗原结合活性验证;采用不同浓度FITC标记的siRNA与scFv-tP进行混合,分别与LiBr细胞(A)及DU145细胞(B)进行混合孵育,观察肿瘤细胞对携载siRNA的单链抗体的内化活性。结果成功构建抗恶性黑素瘤单链抗体-鱼精蛋白片段融合蛋白(Anti-MM scFv-tP)表达载体;实现在大肠杆菌中该融合蛋白的可溶性表达,可获得较纯的scFv-tP蛋白;Anti-MM scFv-tP蛋白与siRNA有明显的结合与携载能力,可以有效与LiBr细胞表面特异性抗原进行结合,且有较强的内化活性。结论本研究构建的抗MM单链抗体-鱼精蛋白融合蛋白表达载体可稳定表达,Anti-MM scFv-tP蛋白具有siRNA结合携载能力,可靶向识别结合进入LiBr细胞,为后续进一步研究该单链抗体融合蛋白靶向载体携载siRNA在体内外抑制恶性黑色素瘤恶性进展奠定坚实的基础。
[Abstract]:Objective to construct the expression vector of anti-MM single-chain antibody-protamine fusion protein and to verify its expression efficiency and function. Methods the truncated fragment of protamine was linked to the single chain antibody gene against melanoma cell surface antigen by PCR, and the fusion protein amplification product was constructed by Taka Rap MD19-T vector kit. The expression vector of GST fusion protein was constructed to realize the soluble expression of the fusion protein in Escherichia coli, and the scFv-tP protein was obtained after two-step affinity purification, and the binding activity of Anti-MM scFv-tP to siRNA was detected by EMSA. Fluorescent molecular markers of scFv-tP protein were detected by flow cytometry and confocal microscopy, respectively, and the cell surface antigen binding activity of scFv-tP was detected by confocal microscopy, and then mixed with scFv-tP with different concentrations of FITC-labeled siRNA. The tumor cells were incubated with libr cells (A) and DU145 cells (B), respectively, to observe the internalization activity of tumor cells against siRNA carrying scFv. Results the fusion protein Anti-MM scFv-tP was successfully constructed and the soluble expression of the fusion protein was realized in Escherichia coli. The purified scFv-tP protein anti-MM scFv-tP protein has obvious binding and carrying ability with siRNA. It can effectively bind to libr cell surface specific antigen and has strong internalization activity. Conclusion the expression vector of anti-MM single-chain antibody and protamine fusion protein constructed in this study can stably express Anti-MM scFv-tP protein with siRNA binding ability and can be targeted recognition binding into libr cells. The results provide a solid foundation for further study on the inhibition of malignant melanoma by siRNA carried by the fusion protein vector in vivo and in vitro.
【作者单位】：西安交通大学第二附属医院皮肤科;河北工程大学附属医院预防保健科;浙江加州国际纳米技术研究院;西安交通大学第一附属医院泌尿外科;西安交通大学基础医学院病原生物学与免疫学系;中国人民解放军第四军医大学实验动物中心;中国军事医学科学院输血研究所;
【基金】：国家自然科学基金(30960349)~~
【分类号】：R739.5

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