ACTA VETERINARIA ET ZOOTECHNICA SINICA ›› 2019, Vol. 50 ›› Issue (1): 126-133.doi: 10.11843/j.issn.0366-6964.2019.01.015

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Characterization of the Interaction between Newcastle Disease Virus Matrix Protein and Chicken Importin β1 Protein by GST Pull-down Assay

HU Yan1, DUAN Zhiqiang1,2*, JI Xinqin1,2, ZHAO Jiafu1,2, DENG Shanshan1, LI Shijing3, XIONG Jianmin1   

  1. 1. College of Animal Science, Guizhou University, Guiyang 550025, China;
    2. Key Laboratory of Animal Genetics, Breeding and Reproduction in the Plateau Mountainous Region, Ministry of Education, Guizhou University, Guiyang 550025, China;
    3. Bureau of Agriculture, Huaxi District, Guiyang 550025, China
  • Received:2018-04-11 Online:2019-01-23 Published:2019-01-24

Abstract:

The purpose of this study was to verify the in vitro interaction between Newcastle disease virus (NDV) matrix (M) protein and chicken importin β1 protein. The products of NDV M gene and chicken importin β1 gene were obtained by PCR amplification, and then subcloned into the prokaryotic expression vectors pGEX-6p-1 and pET-32a(+) to construct plasmids pGEX-6p-M and pET-32a-importin β1, respectively. The recombinant proteins were expressed by transforming the plasmids into BL21(DE3) under the condition of IPTG and then analyzed by SDS-PAGE. The obtained inclusion body weight histones were dealt with through denaturation and renaturation to recover activity. Then the interaction between GST-M (bait protein) and His-importin β1 (prey protein) was examined by GST pull-down assay. The results showed that the recombinant prokaryotic expression vectors pGEX-6p-M and pET-32a-importin β1 were successfully constructed, and the recombinant proteins were correctly expressed in BL21 (DE3). However, GST-M existed in the form of inclusion bodies, but His-importin β1 existed in the form of both solubility and inclusion bodies. The activated GST-M recombinant protein was recovered by protein refolding kit and could capture the His-importin β1 protein when used as bait protein, while GST alone could not. The in vitro interaction between NDV M protein and chicken importin β1 protein was verified by GST pull-down assay, which will provide foundation for further studying the role of importin β1 in the nuclear localization of NDV M protein and the replication and pathogenicity of NDV.

Key words: Newcastle disease virus, matrix protein, importin β1 protein, GST pull-down, protein-protein interaction

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