免疫共沉淀筛选细胞内与A型流感病毒M2蛋白相互作用的蛋白

Screening cellular proteins interacted with M2 protein of influenza A virus by Coimmunoprecipitation

摘要:

摘要：【目的】筛选细胞内与A型流感病毒M2蛋白（A/M2）相互作用的蛋白质。【方法】将A/M2编码序列插入真核表达载体pCAGGS-CFlag，重组质粒pCAGGS-CFlag-A/M2转染HEK-293T细胞，裂解细胞，以Flag单抗偶联的琼脂糖球珠免疫沉淀A/M2-Flag蛋白，清洗去除非特异性结合的杂蛋白后，SDS-PAGE银染法显示与A/M2共沉淀的蛋白，从胶上切下此蛋白条带进行质谱分析。【结果】成功构建了A/M2的表达质粒，免疫印迹证实了A/M2蛋白在293T细胞中能够表达，免疫共沉淀筛选到与A/M2结合的多种蛋白，分析质谱结果，确定ataxin 10和3个真核翻译起始因子(eIF)为候选蛋白。【结论】ataxin 10与A/M2相互作用为流感病毒感染或接种流感疫苗引发小脑性共济失调提供了解释，eIF与A/M2相互作用表明A/M2可能在调控病毒蛋白合成方面起重要作用。

Abstract:

Abstract: [Objective] To screen cellular protein interacted with influenza A M2 protein(A/M2). [Methods] we cloned A/M2 gene fragment into pCAGGS-CFlag vector, and the resulting plasmid was transfected into human embryonic kidney (HEK) 293T cells.The recombinant Flag fusion protein,A/M2-Flag was absorbed specificly by Anti-Flag Monoclonal Antibody M2-Conjugated Agarose beads, we loaded the beads on 12% SDS-PAGE after we washed it with lysis buffer. Silver staining of the gel revealed that several proteins were co-purified with A/M2.To identify the proteins,we excised the protein bands and analysed them by mass spectroscopic sequencing. [Results] We got two kinds of proteins, ataxin 10 and eukaryotic initiation factors(eIFs). [Conclusion] Interaction between Ataxin 10 and A/M2 would explain why inflenza virus infection or influenza vaccine innoculation causes acute cerebellar ataxia. A/M2 interacting with eIFs would imply that A/M2 is involved in the regulation of influenza virus protein synthesis.