鸡传染性支气管炎病毒SD03株N基因的克隆、序列分析及N蛋白理化性质分析

›› 2014, Vol. 41 ›› Issue (12): 67-73.

鸡传染性支气管炎病毒SD03株N基因的克隆、序列分析及N蛋白理化性质分析

邢婧珉¹, 陈冰¹, 李云霞¹, 付立辉², 张立霞¹, 李亚杰¹, 郁宏伟¹, 杨保收¹

1. 天津瑞普生物技术股份有限公司瑞普研究院生物制品研究中心, 天津 300308;
2. 瑞普(天津)生物药业有限公司, 天津 300300

收稿日期:2014-07-04 出版日期:2014-12-20 发布日期:2014-12-25
通讯作者: 郁宏伟 E-mail:yuhw@ringpu.com
作者简介:邢婧珉(1985－),女,内蒙古人,硕士,研究方向:微生物分子生物学与动物疫苗.
基金资助:
瑞普研究院动物疫病流行病学调查专项基金.

Cloning, Sequence Analysis of N Gene and Physicochemical Properties Analysis of the N Protein of Avian Infectious Bronchitis Virus SD03

XING Jing-min¹, CHEN Bing¹, LI Yun-xia¹, FU Li-hui², ZHANG Li-xia¹, LI Ya-jie¹, YU Hong-wei¹, YANG Bao-shou¹

1. Biological Products Research Center, RingPu Biological Research Institute, Tianjin RingPu Bio-technology Co., Ltd., Tianjin 300308, China;
2. Ringpu (Tianjin) Biological Pharmaceutical Co., Ltd., Tianjin 300300, China

Received:2014-07-04 Online:2014-12-20 Published:2014-12-25

摘要/Abstract

摘要： 试验旨在确定国内鸡肾型传染性支气管炎病毒(IBV)流行毒株核蛋白N与市场上现用疫苗株、近年国内外分离株的差异性.本研究利用自山东某鸡场病鸡肾脏组织分离获得、经PCR鉴定为IBV阳性的毒株(命名为SD03株),参考NCBI上部分IBV毒株N基因的保守区域设计引物,进行RT-PCR扩增,构建重组pMD18-T-N/DH5α菌,测序获得N基因序列.与参考株进行了核苷酸及氨基酸同源性分析,结合DNAStar软件与http://www.expasy.org/网站对该基因编码的蛋白进行理化性质分析.结果显示,SD03株为IBV肾型毒株,与LDT3、partridge/GD/S14/2003毒株(肾型)核苷酸及氨基酸同源性均在99.0%以上.与其他国内外肾型经典毒株Ma5、W93、Gray、Holte株氨基酸同源性为90.0%～94.6%,与国内外呼吸型疫苗株H120、H52、M41氨基酸同源性为89.7%～91.0%.本研究深入分析了当前流行株SD03株N蛋白的理化特性,为N蛋白亚单位疫苗在不同系统中表达及蛋白纯化提供了理论依据,为中国现阶段IBV的防控奠定了一定的理论基础.

关键词: 传染性支气管炎病毒; RT-PCR; N基因; N蛋白; 同源性; 系统发生树; N蛋白理化性质

Abstract: The study was aimed to determine the differences between the N nucleoprotein of domestic pandemic kidney type avian infectious bronchitis virus (IBV) strains and other strains such as the ones already existed on the market and the abroad ones isolated in rencent years. Biological products research center of Tianjin RingPu bio-technology Co., Ltd. isolated a strain of new virus in renal tissue of chickens obtained from Shandong farm. It was finally identified to be a positive one belonging to IBV by PCR and named SD03. The primers were designed according to the conserved regions from NCBI. Then constructed the recombinant bacteria named pMD18-T-N/DH5α by RT-PCR and got the gene sequences of N domain through sequencing. The strain of SD03 was identified as kidney type IBV strain, and its homologies of nucleotide and amino acid to LDT3 and partridge/GD/S14/2003 strians (kideny type) were > 99.0% and its homologies of amino acid were 90.0% to 94.6% compared to classical kidney type strains such as Ma5, W93, Gray and Holte strains. While compared to the respiratory vaccine strains (H120, H52, M41) the homologies of amino acid were 89.7% to 91.0%. This research deeply analyzed the physicochemical properties of N domain protein of current reference epidemic strains through DNAStar software and ExPASY website (http://www.expasy.org/). This study provided a theoretical reference for N protein subunit vaccines expression and purification in different systems, and also laid some theoretical foundation for the prevention of IBV.

Key words: infectious bronchitis virus; RT-PCR; N gene; N protein; homology; phylogenetic tree; physicochemical properties of N protein

中图分类号:

邢婧珉, 陈冰, 李云霞, 付立辉, 张立霞, 李亚杰, 郁宏伟, 杨保收. 鸡传染性支气管炎病毒SD03株N基因的克隆、序列分析及N蛋白理化性质分析[J]. , 2014, 41(12): 67-73.

XING Jing-min, CHEN Bing, LI Yun-xia, FU Li-hui, ZHANG Li-xia, LI Ya-jie, YU Hong-wei, YANG Bao-shou. Cloning, Sequence Analysis of N Gene and Physicochemical Properties Analysis of the N Protein of Avian Infectious Bronchitis Virus SD03[J]. , 2014, 41(12): 67-73.

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