猪繁殖与呼吸综合征病毒FS株Nsp9基因的克隆与序列分析

doi:10.16431/j.cnki.1671-7236.2016.09.005

《中国畜牧兽医》 ›› 2016, Vol. 43 ›› Issue (9): 2249-2256.doi: 10.16431/j.cnki.1671-7236.2016.09.005

猪繁殖与呼吸综合征病毒FS株Nsp9基因的克隆与序列分析

赵孟孟^1,2, 宋中宝³, 冯松林¹, 王文佳⁴, 邢星⁵, 冯嘉萍¹, 张桂红¹

1. 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;
2. 河南农业大学牧医工程学院, 郑州 450002;
3. 南京农业大学动物医学院, 南京 210095;
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:2016-02-15 出版日期:2016-09-20 发布日期:2016-09-20
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（CARS-36）项目；国家重点研发计划课题（2016YFD0500707）

Cloning and Sequence Analysis of Nsp9 Gene of Porcine Reproductive and Respiratory Syndrome Virus FS Strain

ZHAO Meng-meng^1,2, SONG Zhong-bao³, FENG Song-lin¹, WANG Wen-jia⁴, XING Xing⁵, FENG Jia-ping¹, ZHANG Gui-hong¹

1. Key Laboratory of Zoonosis Prevention and Control of Guangdong Province, National Engineering Research Center for Breeing Swine Industry, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China;
2. College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou 450002, China;
3. College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, 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:2016-02-15 Online:2016-09-20 Published:2016-09-20

摘要/Abstract

摘要：

为了对猪繁殖与呼吸综合征病毒（PRRSV）Nsp9基因的功能进行预测，利用RT-PCR法从细胞毒中扩增PRRSV FS株的Nsp9基因，并将其克隆至pMD18-T载体上，测序得到Nsp9基因序列，利用多种生物信息学软件分析Nsp9序列生物学信息。结果显示，Nsp9基因全长1 929 bp，目的基因的蛋白分子质量为70.5 ku，pI为7.78，表明该蛋白不稳定；抗原性分析显示，Nsp9基因存在多个抗原位点，柔韧性较好，多处位点的亲水性较强，适合作为单抗制备的表位；同种亚型不同毒株的Nsp9基因氨基酸同源性为96.9%～98.9%，这表明Nsp9基因高度保守，可进一步作为检测靶蛋白用于猪繁殖与呼吸综合征（PRRS）的诊断与疫苗免疫；亚细胞定位预测该基因可能定位于细胞质中，而不是细胞核内；同源建模预测三级结构结果显示，三维结构呈右手型，具有3个亚结构域，没有信号肽。此外，亲本株Nsp9基因与疫苗株Nsp9基因存在多个氨基酸的突变，这些氨基酸的插入与缺失是否与病毒的聚合酶活性和毒力有关还需要进一步试验验证。

关键词: 猪繁殖与呼吸综合征病毒; Nsp9基因; 克隆; 序列分析

Abstract:

In order to evaluate the function of Nsp9 gene,the target gene of porcine reproductive and respiratory syndrome virus (PRRSV) FS strain was amplificated and sequenced after being cloned into the pMD18-T vector.The physical and chemical properties,homology,hydrophilicity,surface probability plot,antigenic index,secondary structure and subcellular localization were predicted by various softwares.The results showed that the length of Nsp9 was 1 929 bp,its predicted molecular weight was 70.5 ku and pI was 7.78,and it was unstable protein.There were many antigen sites,and the flexibility and hydrophilicity of Nsp9 were ideal.The study showed that Nsp9 possessed potential antigenicity,and it fits for preparation of monoclonal antibodies.The results of subcellular location showed that it might exist in the cytoplasm.Nsp9 of FS strain shared 96.9% to 98.9% amino acid homology with other strains of the some hypotype.Some amino acid mutations were found between the parent strains and vaccine strains,and insertions and deletions could be found among the European strains and the American strains.Whether these insertions and deletions correlate with the virulence and polymerase needed further research.

Key words: porcine reproductive and respiratory syndrome virus; Nsp9 gene; clone; sequence analysis

中图分类号:

Q785

赵孟孟, 宋中宝, 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FS株Nsp9基因的克隆与序列分析[J]. 《中国畜牧兽医》, 2016, 43(9): 2249-2256.

ZHAO Meng-meng, SONG Zhong-bao, FENG Song-lin, WANG Wen-jia, XING Xing, FENG Jia-ping, ZHANG Gui-hong. Cloning and Sequence Analysis of Nsp9 Gene of Porcine Reproductive and Respiratory Syndrome Virus FS Strain[J]. , 2016, 43(9): 2249-2256.

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猪繁殖与呼吸综合征病毒FS株Nsp9基因的克隆与序列分析

Cloning and Sequence Analysis of Nsp9 Gene of Porcine Reproductive and Respiratory Syndrome Virus FS Strain

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