鸭坦布苏病毒NS3蛋白的原核表达及其多克隆抗血清的制备

doi:10.16036/j.issn.1000-2650.2020.05.016

四川农业大学学报 ›› 2020, Vol. 38 ›› Issue (05): 619-623.doi: 10.16036/j.issn.1000-2650.2020.05.016

鸭坦布苏病毒NS3蛋白的原核表达及其多克隆抗血清的制备

林霄¹, 贺煜¹, 程安春^1,2,3, 汪铭书^1,2,3, 贾仁勇^1,2,3, 朱德康^{2, 3}, 刘马峰¹, 杨乔^1,2,3, 吴英^1,2,3, 赵新新^1,2,3, 张沙秋^1,2,3, 黄娟^1,2,3, 刘韵雅^1,2,3, 张玲^1,2,3, 潘磊昌^1,2,3, 欧旭敏^1,2,3, 毛塞^1,2,3, 陈舜^1,2,3,*

1.四川农业大学动物医学院禽病防治研究中心,成都 611130;
2.四川农业大学动物医学院预防兽医研究所,成都 611130;
3.动物疾病与人类健康四川省重点实验室,成都 611130

收稿日期:2020-06-05 出版日期:2020-10-28 发布日期:2021-02-05
作者简介:林霄,硕士研究生。^*责任作者：陈舜,博士,教授,主要从事禽病病原学研究,E-mail：shunchen@sicau.edu.cn。
基金资助:
四川省重点研发计划项目（2018NZ0005）; 国家重点研发计划项目（2017YFD0500800）

Prokaryotic Expression of Duck Tembusu Virus NS3 and Its Polyclonal Antiserum Preparation

LIN Xiao¹, HE Yu¹, CHENG Anchun^1,2,3, WANG Mingshu^1,2,3, JIA Renyong^1,2,3, ZHU Dekang^{2, 3}, LIU Mafeng¹, YANG Qiao^1,2,3, WU Ying^1,2,3, ZHAO Xinxin^1,2,3, ZHANG Shaqiu^1,2,3, HUANG Juan^1,2,3, LIU Yunya^1,2,3, ZHANG Ling^1,2,3, PAN Leichang^1,2,3, OU Xumin^1,2,3, MAO Sai^1,2,3, CHEN Shun^1,2,3,*

1. Institute of Preventive Veterinary Medicine,Sichuan Agricultural University,Chengdu 611130,China;
2. Key Laboratory of Animal Disease and Human Health of Sichuan Province,Chengdu 611130,China;
3. Avian Disease Research Center,College of Veterinary Medicine of Sichuan Agricultural University, Chengdu 611130,China

Received:2020-06-05 Online:2020-10-28 Published:2021-02-05

摘要/Abstract

摘要： 【目的】利用原核表达系统表达鸭坦布苏病毒（Duck Tembusu virus,DTMUV）的NS3蛋白,并以此制备鼠抗NS3蛋白的多克隆抗血清。【方法】利用PCR扩增鸭坦布苏病毒CQW1株NS3基因片段,然后将其克隆到pET-28a(+)载体,构建重组质粒pET-28a(+)-NS3。将重组表达质粒转化到大肠杆菌BL21感受态细胞中,筛选获得阳性重组菌,经IPTG诱导表达,通过SDS-PAGE和Western blot试验表明重组NS3蛋白在BL21细胞中成功表达,将重组NS3蛋白切胶纯化后免疫小鼠后收获抗血清。【结果】经间接免疫荧光试验和Western blot试验验证,多克隆抗血清可识别病毒感染细胞过程产生的NS3蛋白和真核表达的NS3蛋白,具有良好的反应性和特异性。【结论】成功制备的DTMUV NS3蛋白的鼠源多克隆血清,将为鸭坦布苏病毒的临床检测和NS3蛋白功能性研究奠定基础。

关键词: 鸭坦布苏病毒, NS3蛋白, 原核表达, 多抗血清

Abstract: 【Objective】 In this study, NS3 protein of Duck Tembusu virus (DTMUV) was expressed usingprokaryotic expression system, and subsequently used to prepare polyclonal anti-serum of mouse anti-NS3 protein. 【Method】 NS3 gene fragment of CQW1 strain of DTMUV was amplified by PCR, and then cloned into pET-28a (+) vector to construct recombinant plasmid pET-28a (+) -NS3. The recombinant expression plasmid was transformed into Escherichia coli BL21 competent cells, and the positive recombinant bacteria were screened and expressed by IPTG induction. The results based on SDS-PAGE and Western blot showed that the recombinant NS3 protein was successfully expressed in BL21 cells. The recombinant NS3 protein was purified and used to immunize mice, the antiserum was harvested. 【Result】 The indirect immunofluorescence assay and Western blot assay confirmed that the polyclonal antiserum can recognize the NS3 protein produced by the DTMUV-infected cells and the NS3 protein expressed by eukaryotic cells, which has good reactivity and specificity. 【Conclusion】 The mouse polyclonal sera of DTMUV NS3 protein successfully prepared in this study, and it will lay the foundation for the clinical detection of DTMUV and the functional study of NS3 protein.

Key words: duck tembusu virus, NS3 protein, prokaryotic expression, polyclonal antiserum

中图分类号:

S852.65

林霄, 贺煜, 程安春, 汪铭书, 贾仁勇, 朱德康, 刘马峰, 杨乔, 吴英, 赵新新, 张沙秋, 黄娟, 刘韵雅, 张玲, 潘磊昌, 欧旭敏, 毛塞, 陈舜. 鸭坦布苏病毒NS3蛋白的原核表达及其多克隆抗血清的制备[J]. 四川农业大学学报, 2020, 38(05): 619-623.

LIN Xiao, HE Yu, CHENG Anchun, WANG Mingshu, JIA Renyong, ZHU Dekang, LIU Mafeng, YANG Qiao, WU Ying, ZHAO Xinxin, ZHANG Shaqiu, HUANG Juan, LIU Yunya, ZHANG Ling, PAN Leichang, OU Xumin, MAO Sai, CHEN Shun. Prokaryotic Expression of Duck Tembusu Virus NS3 and Its Polyclonal Antiserum Preparation[J]. Journal of Sichuan Agricultural University, 2020, 38(05): 619-623.

参考文献

[1] 朱丽萍, 颜世敢. 鸭坦布苏病毒研究进展[J]. 中国预防兽医学报, 2012, 34(1): 79-82.
[2] SU J L, LI S, HU X D, et al.Duck egg-drop syndrome caused by BYD Virus, a new tembusu-related flavivirus[J]. PLoS ONE, 2011, 6(3): e18106.
[3] SUN X L, DIAO Y X, WANG J L, et al.Tembusu virus infec-tion in Cherry Valley ducks: The effect of age at infection[J]. Vet Microbiol, 2014, 168(1): 16-24.
[4] CAO Z Z, ZHANG C, LIU Y H, et al.Tembusu virus in ducks, China[J]. Emerg Infect Dis, 2011, 17(10): 1873-1875.
[5] ZHANG W, CHEN S, MAHALINGAM S, et al.An updated review of avian-origin Tembusu virus: a newly emerging avian Flavivirus[J]. Journal of General Virology, 2017, 98(10): 2413-2420.
[6] WANG H J, LI X F, LIU L, et al.The emerging duck flaviv-irus is not pathogenic for primates and is highly densitive to mammalian interferon sntiviral signaling[J]. Journal of General Virology, 2016, 90(14): 6538-6548.
[7] 周晓雅, 周祺, 殷冬冬, 等. 鸭坦布苏病毒感染对BHK-21细胞分泌胞外体的影响[J]. 中国免疫学杂志, 2018, 34(2): 161-166.
[8] 陈浩. 坦布苏病毒ZC株感染性克隆的构建和部分3’非编码区功能的初步研究[D]. 泰安: 山东农业大学, 2015.
[9] HE Y, WANG A Q, CHEN S, et al.Differential immune-related gene expression in the spleens of duck Tembusu virus-infected goslings[J]. Vet Microbiol, 2017, 212: 39-47.
[10] CHEN S, HE Y, ZHANG R J, et al.Establishment of a reverse genetics system for duck Tembusu virus to study virulence and screen antiviral genes[J]. Antiviral Research, 2018, 157: 120-127.
[11] FALGOUT B, PETHEL M, ZHANG Y M, et al.Both nonstru-ctural proteins NS2B and NS3 are required for the proteolytic processing of dengue virus nonstructural proteins[J]. Journal of Virology, 1991, 65(5): 2467-2475.
[12] YON C, TERAMOTO T, MUELLER N, et al.Modulation of the nucleoside triphosphatase/RNA helicase and 5′-RNA triphos-phatase activities of dengue virus type 2 nonstructural protein 3(NS3) by interaction with NS5, the RNA-dependent RNA polymerase[J]. Journal of Biological Chemistry, 2005, 280:27412-27419.
[13] LUO D, XU T, WATSON R P, et al.Insights into RNA unwi-nding and ATP hydrolysis by the flavivirus NS3 protein[J]. Embo Journal, 2008, 27(23): 3209-3219.
[14] 汪志铮. 鸭坦布苏病毒病简介[J]. 科学种养,2018(7): 52.
[15] 陈政. 乙型脑炎病毒NS3蛋白与宿主蛋白相互作用的研究[D]. 武汉: 华中农业大学, 2016.
[16] VALERIE K, RADHAKRISHNAN P, KYUNG C.Flaviviral replication complex: coordination between RNA synthesis and 5’-RNA capping[J]. Viruses, 2015, 7(8): 4640-4656.
[17] 石迎. 鸭坦布苏病毒反向遗传操作系统的建立[D]. 泰安: 山东农业大学, 2014.

鸭坦布苏病毒NS3蛋白的原核表达及其多克隆抗血清的制备

Prokaryotic Expression of Duck Tembusu Virus NS3 and Its Polyclonal Antiserum Preparation

PDF (PC)

赞

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 9

Metrics

本文评价

推荐阅读 10

[1]	刘裕峰, 朱天辉, 刘应高, 谯天敏, 李姝江, 龙旭梅, 韩珊. 板栗过氧化氢酶基因CmCAT的克隆与原核表达[J]. 四川农业大学学报, 2019, 37(02): 168-176.
[2]	阳明贤, 左之才, 李碧, 王宇. 牛抵抗素克隆及高效可溶性表达研究[J]. 四川农业大学学报, 2018, 36(06): 802-807.
[3]	李青青, 张润敏, 石冠蓝, 姚攀锋, 王晓丽, 李成磊. 苦荞黄酮醇合酶基因FtFLS4的克隆及其在大肠杆菌的表达[J]. 四川农业大学学报, 2018, 36(05): 611-617.
[4]	张烨, 赵术珍, 王江山, 夏晗, 侯蕾, 任丽, 王兴军. 花生光敏色素AhphyA和AhphyB基因的克隆及原核表达研究[J]. 四川农业大学学报, 2015, 33(01): 13-21.
[5]	徐黎黎, 钟美玉, 李伟, 魏育明. 阿拉拉特小麦高分子量谷蛋白新基因1Gy的克隆及序列分析[J]. 四川农业大学学报, 2011, 29(02): 141-146.
[6]	刘菲, 高丽芹, 程安春, 汪铭书, 韩清林, 赵婷婷, 唐卫杰, 严晓玲. 天府肉鹅IFN-α成熟肽基因的原核表达及抗病毒活性[J]. 四川农业大学学报, 2011, 29(02): 269-273.
[7]	李安明, 邓青云, 李德华. 长喙田菁植物螯合肽合成酶PCS1的原核表达及纯化[J]. 四川农业大学学报, 2011, 29(02): 213-217.
[8]	陈红英, 崔沛, 崔保安, 胡功政, 朱前磊, 王子馨. 罗曼鸡白细胞介素18基因的克隆、表达及生物学活性检测[J]. 四川农业大学学报, 2009, 27(02): 218-222.
[9]	宋振辉, 郭万柱, 韩国全, 骆爱芳. 猪传染性胃肠炎病毒SC-Y株N基因的克隆及原核表达[J]. 四川农业大学学报, 2006, 24(02): 210-213.