摘要
【目的】为探究H7N9亚型禽流感病毒(AIV)PA-X蛋白的功能,采用GST pull-down联合质谱技术筛选与鉴定与PA-X相互作用的宿主蛋白。【方法】构建PA-X的重组表达载体pGEX-4T-PA-X,转化大肠杆菌BL21(DE3)中表达重组蛋白,并利用GST亲合树脂进行纯化得到GST-PA-X融合蛋白。利用GST pull-down技术,将GST-PA-X和GST对照蛋白分别与A549细胞裂解液总蛋白进行体外孵育,得到的蛋白复合物经SDS-PAGE分离后进行质谱检测分析。【结果】经GST pull-down筛选与质谱分析鉴定后得到39个高可信度的可能与PA-X互作的候选蛋白质。通过GO注释和KEGG等通路富集分析发现,这些蛋白主要参与RNA的代谢及加工、mRNA翻译及运输、多肽生物合成及基因表达的转录后调节等生物学过程。对其中评分最高的蛋白Hsp70-2与PA-X的相互作用作进一步验证,免疫荧光试验结果表明Hsp70-2与PA-X可以在细胞浆中共定位。【结论】利用GST pulldown联合质谱技术筛选到39种与PA-X互作的候选蛋白,且经免疫荧光试验证明候选蛋白Hsp70-2与PA-X存在共定位,为深入探讨PA-X在AIV生命周期中的作用机制奠定基础。
【Objective】In order to explore the function of PA-X protein from H7 N9 avian influenza virus(AIV),the host proteins interacted with PA-X were screened and identified by GST pull-down and mass spectrometry technology.【Method】The recombinant expression vector pGEX-4 T-PA-X was constructed and transformed into Escherichia coli BL21(DE3)cells to express the recombinant protein,and GST-PA-X fusion protein was purified with GST affinity resin.With GST pull-down technique,GST-PA-X and GST control protein were respectively incubated with total proteins from the A549 cell lysate in vitro,the protein complexes were separated by SDS-PAGE and then analyzed by mass spectrometry.【Result】The mass spectrometry analysis demonstrated that 39 candidate proteins with a high credibility might interact with PA-X.Through the enrichment analysis of GO and KEGG pathway,it was found that these proteins were mainly involved in biological processes such as RNA metabolism and processing,mRNA translation and transport,peptide biosynthetic process,and posttranscriptional regulation of gene expression and so on.As one of the key candidate proteins,Hsp70-2 was further chosen to explore its interaction with PA-X.The immunofluorescent analysis demonstrated that Hsp70-2 and PA-X co-localized in the cytoplasm of the A549 cell line.This further confirmed the interaction between cellular Hsp70-2 and PA-X.【Conclusion】Thirty-nine candidate proteins were identified to interact with PA-X in A549 cells via the GST pull-down combined with mass spectrometry technology,and the colocalization between Hsp70-2 and PA-X was confirmed by the immunofluorescence test.These results provide a basis for a further research on the role of PA-X in the life cycle of AIV.
作者
徐国双
姜童潇
郭艺迪
段铭
张茂林
关振宏
XU Guoshuang;JIANG Tongxiao;GUO Yidi;DUAN Ming;ZHANG Maolin;GUAN Zhenhong(Institute of Zoonosis,Jilin University/Key Laboratory of Zoonosis,Ministry of Education,Changchun 130062,China)
出处
《广东农业科学》
CAS
2022年第1期121-127,共7页
Guangdong Agricultural Sciences
基金
国家自然科学基金(32072902)。
