Lin Cui, Tang Wen, Gu Jinyan, Jin Yulan, Dong Weiren, Liao Min, Zhou Jiyong
Key Laboratory of Animal Virology of Ministry of Agriculture, College of Animal Sciences, Zhejiang University, Hangzhou 310058, Zhejiang, China.
Institute of Immunology, Nanjing Agricultural University, Nanjing 210000, Jiangsu, China.
Sheng Wu Gong Cheng Xue Bao. 2019 Jan 25;35(1):40-48. doi: 10.13345/j.cjb.180098.
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.
最近在猪圆环病毒2型（PCV2）中发现了一种由开放阅读框4（ORF4）编码的新型蛋白质。然而，关于ORF4的相互作用蛋白知之甚少，这阻碍了对ORF4在PCV2生命周期中的生物学功能的更好理解。在本研究中，将ORF4插入pCMV-N-Flag-GST的多克隆位点，产生重组质粒pCMV-N-Flag-GST-ORF4。将重组质粒转染到293T细胞中，通过GST下拉和SDS-PAGE依次富集和分离ORF4的细胞内相互作用复合物。用银染法对PCV2 ORF4的潜在相互作用蛋白进行染色，并通过质谱（MS）进行鉴定。最后，鉴定出了5种与ORF4相互作用的候选蛋白，包括丝氨酸/苏氨酸蛋白磷酸酶6催化亚基、α心肌1、肌动蛋白、SEC14样蛋白5和肌球蛋白9。这些结果将有助于更好地理解ORF4在PCV2感染细胞中的生物学功能。
