GST pull-down结合质谱筛选猪圆环病毒2型ORF4潜在互作蛋白

Combination of mass spectrometry and GST pull-down techniques to study potential interacting protein of PCV2 ORF4

摘要:

猪圆环病毒2型编码的ORF4蛋白是近年来发现的新蛋白。迄今为止，人们对ORF4所参与的细胞生物学过程知之甚少。本研究首先构建了带双标签的真核表达载体pCMV-N-Flag-GST，再将ORF4基因插入该载体中，形成pCMV-N-Flag-GST-ORF4。将质粒转染293T细胞表达ORF4后，通过GST Pull-down试验捕获细胞内潜在与ORF4互作的蛋白库。经SDS-PAGE分离及银染后，对所得的特异性条带进行质谱鉴定，筛选出5个与ORF4潜在互作的蛋白，包括丝氨酸/苏氨酸蛋白磷酸酶6催化亚基、α心肌蛋白、β肌动蛋白、SEC-14样蛋白5和肌球蛋白myosin 9。上述研究结果为深入揭示ORF4在病毒感染细胞过程中发挥的作用提供新的思路与方向。

Abstract:

A novel protein encoded by the open reading frame 4 (ORF4) was recently discovered in porcine circovirus type 2 (PCV2). However, little is known about the interaction proteins of ORF4 which hindered better understanding the biological functions of ORF4 in the life cycle of PCV2. In the present study, the ORF4 was inserted into the multiple cloning site of pCMV-N-Flag-GST, yielding recombinant plasmid pCMV-N-Flag-GST-ORF4. The recombinant plasmid was transfected into 293T cells and the intracellular interaction complex of ORF4 were enriched and separated by GST pull-down and SDS-PAGE, sequentially. The potential interacting proteins of PCV2 ORF4 were stained with silver and identified by mass spectrometry (MS). Finally, five candidate ORF4-interacting proteins, including Serine/threonine-protein phosphatase 6 catalytic subunit, alpha cardiac muscle 1, actin, SEC14-like protein 5 and myosin 9 were identified. These results would benefit a better understanding of the biological function of ORF4 in PCV2 infected cells.