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《Chinese Journal of Animal and Veterinary Sciences》 2019-01
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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 Yan;DUAN Zhiqiang;JI Xinqin;ZHAO Jiafu;DENG Shanshan;LI Shijing;XIONG Jianmin;College of Animal Science,Guizhou University;Key Laboratory of Animal Genetics,Breeding and Reproduction in the Plateau Mountainous Region,Ministry of Education,Guizhou University;Bureau of Agriculture,Huaxi District;  
The purpose of this study was to verify the in vitrointeraction 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-1and 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 Hisimportin 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 vitrointeraction 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.
【Fund】: 国家自然科学基金(31502074;31760732);; 贵州省科技计划项目(黔科合平台人才[2017]5788号);; 贵州省农业攻关项目(黔科合支撑[2016]2588号);; 贵州省科学技术基金(黔科合J字[2015]2054号)
【CateGory Index】: S852.65
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