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

Cui Lin; Wen Tang; Jinyan GU; Yulan Jin; Weiren Dong; Min Liao; Jiyong Zhou

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

Author: Cui LIN ¹ ; Wen TANG ¹ ; Jinyan GU ¹ ; Yulan JIN ¹ ; Weiren DONG ¹ ; Min LIAO ¹ ; Jiyong ZHOU ¹
Author Information

1. Key Laboratory of Animal Virology of Ministry of Agriculture, College of Animal Sciences, Zhejiang University, Hangzhou 310058, Zhejiang, China.
Publication Type:Journal Article
Keywords: GST pull-down; ORF4; interacting protein; porcine circovirus type 2
MeSH: Animals; Circoviridae Infections; Circovirus; HEK293 Cells; Humans; Mass Spectrometry; Open Reading Frames; Swine; Viral Proteins
From: Chinese Journal of Biotechnology 2019;35(1):40-48
CountryChina
Language:Chinese
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.