猪繁殖与呼吸综合征病毒强、弱毒株Nsp9基因的反向遗传学替换改造

doi:10.16431/j.cnki.1671-7236.2016.11.007

《中国畜牧兽医》 ›› 2016, Vol. 43 ›› Issue (11): 2852-2858.doi: 10.16431/j.cnki.1671-7236.2016.11.007

猪繁殖与呼吸综合征病毒强、弱毒株Nsp9基因的反向遗传学替换改造

赵孟孟^1,2, 钱娟娟³, 崔甜甜², 冯松林², 王文佳⁴, 邢星⁵, 冯嘉萍², 张桂红²

1. 河南农业大学牧医工程学院, 郑州 450002;
2. 华南农业大学兽医学院, 国家生猪种业工程技术研究中心, 广东省动物源性人兽共患病预防与控制重点实验室, 广州 510642;
3. 中国科学院动物研究所膜生物学国家重点实验室, 北京 100101;
4. 河南牧业经济学院制药工程学院, 郑州 450046;
5. 广西钦州保税港区出入境检验检疫局, 钦州 535008

收稿日期:2016-02-01 出版日期:2016-11-20 发布日期:2016-11-18
通讯作者: 张桂红 E-mail:guihongzh@scau.edu.cn
作者简介:赵孟孟(1986-),男,河南汝州人,博士,研究方向:猪繁殖与呼吸综合征病毒的致病机理,E-mail:zhaomengmeng502@163.com
基金资助:
国家自然科学基金：猪繁殖与呼吸综合征病毒非结构蛋白Nsp9功能研究项目（31272564）；公益性行业（农业）科研专项经费（201203039）；国家生猪现代农业产业技术体系项目（CARS-36）；国家重点研发计划课题（2016YFD0500707）

Substitution between Virulent and Avirulent PPRSV Vaccine Strains with Reverse Genetics System

ZHAO Meng-meng^1,2, QIAN Juan-juan³, CUI Tian-tian², FENG Song-lin², WANG Wen-jia⁴, XING Xing⁵, FENG Jia-ping², ZHANG Gui-hong²

1. College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou 450002, China;
2. Key Laboratory of Zoonosis Prevention and Control of Guangdong Province, National Engineering Research Center for Breeding Swine Industry, College of Veterinary, South China Agricultural University, Guangzhou 510642, China;
3. State Key Laboratory of Biomembrane and Membrane Biotechnology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China;
4. School of Pharmaceutical Engineering, Henan University of Animal Husbandry and Economy, Zhengzhou 450046, China;
5. Guangxi Qinzhou Free Trade Port Area Entry-exit Inspection and Quarantine Bureau, Qinzhou 535008, China

Received:2016-02-01 Online:2016-11-20 Published:2016-11-18

摘要/Abstract

摘要：

猪繁殖与呼吸综合征病毒（porcine reproductive and respiratory syndrome virus，PRRSV）Nsp9基因编码RNA依赖的RNA聚合酶，在病毒复制中发挥重要作用。为了探索Nsp9基因与病毒的毒力、复制能力及细胞嗜性的相关性，本研究利用反向遗传学技术将PRRSV弱毒疫苗毒CH-1R的Nsp9基因进行克隆，酶切后连接到强毒株XH-GD的感染性克隆上，进行病毒的拯救。结果表明，成功构建替换了CH-1R Nsp9基因的重组病毒。对重组病毒的生物学特性研究发现，拯救病毒的生长速度和病毒滴度明显高于亲本毒株rXH-GD，但是低于疫苗毒CH-1R，重组毒株的噬斑大小与CH-1R相似，初步推测疫苗株CH-1R的聚合酶有助于提高病毒的滴度。本试验为进一步研究Nsp9基因对病毒毒力的影响奠定基础。

关键词: 猪繁殖与呼吸综合征病毒; Nsp9基因; 反向遗传学; 病毒拯救

Abstract:

The Nsp9 gene of porcine reproductive and respiratory syndrome virus（PRRSV）coded the RNA polymerase,which played an important role in the virus replication.In order to determine the interaction between the Nsp9 gene and virulence,replication capacity and cell tropism,the Nsp9 gene sequence of PPRSV vaccine strain CH-1R was cloned,and ligated to a virulent strain XH-GD infectious clone after restriction enzyme digestion,and transformed the virus under the basis of the reverse genetics.The results showed that the virus was successfully saved.And the results of biological characteristics test showed that titers and growth rate of saved virus were obviously higher than that of parental strain rXH-GD,but were lower than vaccine strain CH-1R.While plaques of two strains were similar in size.The polymerase of vaccine strain CH-1R could help to improve the drop degree of the virus,and this test could lay the foundation for further study of the effect of Nsp9 gene on virus virulence.

Key words: PRRSV; Nsp9 gene; reverse genetics; virus salvation

中图分类号:

Q785

赵孟孟, 钱娟娟, 崔甜甜, 冯松林, 王文佳, 邢星, 冯嘉萍, 张桂红. 猪繁殖与呼吸综合征病毒强、弱毒株Nsp9基因的反向遗传学替换改造[J]. 《中国畜牧兽医》, 2016, 43(11): 2852-2858.

ZHAO Meng-meng, QIAN Juan-juan, CUI Tian-tian, FENG Song-lin, WANG Wen-jia, XING Xing, FENG Jia-ping, ZHANG Gui-hong. Substitution between Virulent and Avirulent PPRSV Vaccine Strains with Reverse Genetics System[J]. , 2016, 43(11): 2852-2858.

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猪繁殖与呼吸综合征病毒强、弱毒株Nsp9基因的反向遗传学替换改造

Substitution between Virulent and Avirulent PPRSV Vaccine Strains with Reverse Genetics System

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