J亚群禽白血病病毒gp85蛋白的真核表达纯化及其生物学活性分析

Eukaryotic expression purification and biologic activity analysis of avian leukosis virus gp85

gp85蛋白是禽白血病病毒(ALV)的囊膜表面蛋白,含有病毒-受体决定簇,通过识别和结合受体介导病毒侵入宿主细胞。为表达具有正确构象和生物学活性的J亚群ALV(ALV-J)gp85蛋白并对其生物活性进行分析,本研究以pCAGGS为载体,构建N端带有信号肽编码序列、C端融合Fc编码序列的gp85重组表达质粒pCAGGS-s-gp85-Fc,将其转染293T细胞进行瞬时表达,收集细胞培养液。SDS-PAGE结果表明,gp85-Fc蛋白高效表达,并且经Protein A亲和层析纯化得到高纯度的gp85-Fc蛋白。利用HRV 3C蛋白酶切除Fc标签并且进行分子筛纯化,得到纯度高于90%的gp85蛋白单体。经过流式细胞仪检测,表达的可溶性gp85蛋白能够与ALV-J受体特异性结合。病毒感染阻断试验结果显示,gp85蛋白能够通过封闭受体阻断ALV-J进入DF-1细胞,并且呈现剂量依赖性,在100μg/mL时仍能阻断70%以上的病毒侵入。本研究可溶性的、具有生物学活性的gp85蛋白的制备为体外研究病毒感染宿主细胞机制奠定了的基础。

gp85 is the surface unit （SU） of avian leukosis virus （ALV） envelop which mediates ALV infecting cells by interacting with specific receptor. To express soluble ALV-J gp85 and analysis its biological activity, we constructed a combined gp85 plasmid pCAGGS-s-gp85-Fc that fused a signal peptide coding-sequences onN terminal and Fc coding-sequences on C terminal. After transfected 293T cells with the pCAGGS-s-gp85-Fc plasmid, the supematant was collected and the expression of gp85-Fe was detected with SDS-PAGE. By affinity chromatography of protein A, we obtained gp85-Fc protein with high purity. After cut the Fc tag with HRV 3C protease, gp85 monomer with more than 90% purity was obtained. By flow cytometry detected, we identified that the pure gp85 protein could bind with the receptor specially. Meanwhile, gp85 could block ALV-J enter into DF-1 cells by sealing viral receptor in a dose-dependent manner. When the concentration of gp85 was 100μg/mL, over 70% virus were blocked. These results indicated that soluble gp85 protein was successfully expressed with biologic activity, which would lay the foundation of studying the mechanism of ALV-J infecting cells in vitro.