GST pull-down验证新城疫病毒基质蛋白与鸡importin β1蛋白的相互作用

doi:10.11843/j.issn.0366-6964.2019.01.015

畜牧兽医学报 ›› 2019, Vol. 50 ›› Issue (1): 126-133.doi: 10.11843/j.issn.0366-6964.2019.01.015

GST pull-down验证新城疫病毒基质蛋白与鸡importin β1蛋白的相互作用

胡焱¹, 段志强^1,2*, 嵇辛勤^1,2, 赵佳福^1,2, 邓珊珊¹, 李世静³, 熊建民¹

1. 贵州大学动物科学学院, 贵阳 550025;
2. 贵州大学高原山地动物遗传育种与繁殖教育部重点实验室, 贵阳 550025;
3. 贵阳市花溪区农业局, 贵阳 550025

收稿日期:2018-04-11 出版日期:2019-01-23 发布日期:2019-01-23
通讯作者: 段志强,主要从事家禽抗病育种与疫病防控新技术研究,E-mail:zqduan@gzu.edu.cn
作者简介:胡焱(1992-),男,贵州毕节人,硕士生,主要从事家禽疫病发病机制研究,E-mail:18285127034@163.com
基金资助:
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）

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

HU Yan¹, DUAN Zhiqiang^1,2*, JI Xinqin^1,2, ZHAO Jiafu^1,2, DENG Shanshan¹, LI Shijing³, XIONG Jianmin¹

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:2019-01-23

摘要/Abstract

摘要：

旨在利用GST pull-down技术验证新城疫病毒（Newcastle disease virus，NDV）基质（matrix，M）蛋白与鸡importin β1蛋白在体外的相互作用。根据NDV ZJ1株M基因和鸡importin β1基因序列设计引物，通过PCR扩增获得NDV M基因和鸡importin β1基因，将其分别定向插入到原核表达载体pGEX-6p-1和pET-32a（+）构建重组原核表达载体pGEX-6p-M、pET-32a-importin β1，然后转化至大肠杆菌BL21（DE3）后进行IPTG诱导表达，SDS-PAGE检测重组蛋白的表达情况，并对包涵体重组蛋白进行变性和复性处理。然后以GST-M重组蛋白为诱饵蛋白，His-importin β1重组蛋白为猎物蛋白，利用GST pull-down技术验证M蛋白与importin β1蛋白的相互作用。结果表明，成功构建了重组原核表达载体pGEX-6p-M和pET-32a-importin β1，将其转化至大肠杆菌BL21（DE3）获得了正确表达。SDS-PAGE电泳检测显示GST-M重组蛋白主要以包涵体形式存在，而His-importin β1重组蛋白以可溶性和包涵体两种形式存在。利用蛋白重折叠试剂盒获得了有活性的GST-M重组蛋白，将其作为诱饵蛋白，可以捕获并检测到His-importin β1重组蛋白，但是GST标签蛋白不能结合His-importin β1重组蛋白。利用GST pull-down技术证实了NDV M蛋白与鸡importin β1蛋白在体外具有直接的相互作用，这为深入研究importin β1蛋白在NDV M蛋白细胞核定位的分子机制以及在NDV复制和致病中的作用奠定了基础。

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.

中图分类号:

S855.3

胡焱, 段志强, 嵇辛勤, 赵佳福, 邓珊珊, 李世静, 熊建民. GST pull-down验证新城疫病毒基质蛋白与鸡importin β1蛋白的相互作用[J]. 畜牧兽医学报, 2019, 50(1): 126-133.

HU Yan, DUAN Zhiqiang, JI Xinqin, ZHAO Jiafu, DENG Shanshan, LI Shijing, XIONG Jianmin. Characterization of the Interaction between Newcastle Disease Virus Matrix Protein and Chicken Importin β1 Protein by GST Pull-down Assay[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(1): 126-133.

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GST pull-down验证新城疫病毒基质蛋白与鸡importin β1蛋白的相互作用

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

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