沙粒病毒聚合酶C端的表达纯化与结晶条件筛选 <sup>*</sup>

doi:10.13523/j.cb.20191203

中国生物工程杂志

2019, Vol. 39

Issue (12): 18-23 DOI: 10.13523/j.cb.20191203

研究报告

沙粒病毒聚合酶C端的表达纯化与结晶条件筛选 ^*

景佳美^1,²,徐欣²,王敏¹,彭如超¹,施一^1,^2,^**(

)

1 中国科学院微生物研究所北京 100101
2 中国科学院大学存济医学院北京 100049

Expression and Purification of C-terminal of Arenavirus Polymerase and Screening of Crystallization Conditions

JING Jia-mei^1,²,XUN Xin²,WANG Min¹,PENG Ru-chao¹,SHI Yi^1,^2,^**(

)

1 Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China
2 Savaid Medical School, University of Chinese Academy of Sciences, Beijing 100049, China

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摘要：

拉沙病毒和马秋波病毒同属于沙粒病毒科,人类感染后病死率很高,对人类生命健康造成了严重威胁。沙粒病毒在基因组转录过程中存在与其他分节段负链RNA病毒相似的抢帽机制,位于病毒聚合酶N端的核酸内切酶结构域负责切割宿主mRNA,而其C端负责结合宿主mRNA帽子。目前对哺乳动物沙粒病毒聚合酶C端的特征知之甚少。为此构建了拉沙病毒和马秋波病毒聚合酶C端的重组表达载体,并利用E.coli原核系统进行表达和后续纯化,获得了不同聚体形式的C端蛋白,通过分析超速离心实验和负染电镜观察对其不同的聚体形式进行了表征。最后通过大量筛选结晶条件,得到了拉沙病毒聚合酶C端的晶体,为进一步研究其三维结构和功能机制奠定了基础。

关键词： 拉沙病毒; 马秋波病毒; 聚合酶C端; 表达纯化; 结晶

Abstract:

The Lassa virus and the Machupo virus belong to the family of the Arenaviridae. They can cause human infection with high mortality and pose a threat to the public health. The arenavirus possesses a cap-snatching mechanism for transcription of viral genome, which is similar to that of other segmented negative-strand RNA viruses. This process involves cleavage of host mRNAs by an endonuclease (EN) domain located in the N-terminal region of the viral polymerase, and the cap-binding by the C-terminal region which has not been verified in Mammarenavirus. To this end, the recombinant expression vectors for the C-terminal region of Lassa virus and Machupo virus polymerase were constructed. The recombinant proteins were expressed in E. coli and was found that the proteins can exist with different oligomerization forms in solution.The oligomerization features by negative staining electron microscopy also were characterized. Finally, initial crystals of the C-terminal of Lassa virus polymerase were obtained, which provides a foundation for further study of its three-dimensional structure and functional mechanism.

Key words: Lassa virus Machupo virus C-terminal region of polymerase Expression and purification Crystallization

收稿日期: 2019-04-16 出版日期: 2020-01-15

ZTFLH:

Q819

基金资助: * 中国科学院战略性先导科技专项(B类XDB29010000);国家科技重大专项(2018ZX10101004);国家自然科学基金优秀青年科学基金(81622031);中国科学院青年促进会项目(2015078)

通讯作者: 施一 E-mail: shiyi@im.ac.cn

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引用本文:

景佳美,徐欣,王敏,彭如超,施一. 沙粒病毒聚合酶C端的表达纯化与结晶条件筛选 ^*[J]. 中国生物工程杂志, 2019, 39(12): 18-23.

JING Jia-mei,XUN Xin,WANG Min,PENG Ru-chao,SHI Yi. Expression and Purification of C-terminal of Arenavirus Polymerase and Screening of Crystallization Conditions. China Biotechnology, 2019, 39(12): 18-23.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20191203 或 https://manu60.magtech.com.cn/biotech/CN/Y2019/V39/I12/18

图1 LASV-L-CTD(a)和MACV-L-CTD(b)的凝胶过滤层析和SDS-PAGE图谱

图2 LASV-L-CTD_2(a)、LASV-L-CTD_3(b)和MACV-L-CTD_2(c)的分析超速离心实验结果

图3 LASV-L- CTD_1(a)和MACV-L-CTD_1(b)的多聚体负染电镜照片

图4 LASV-L-CTD的晶体显微照片

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