猫传染性腹膜炎病毒AH1905株N基因的生物信息学分析及原核表达

doi:10.3969/j.issn.1004-1524.2020.03.04

浙江农业学报 ›› 2020, Vol. 32 ›› Issue (3): 406-414.DOI: 10.3969/j.issn.1004-1524.2020.03.04

猫传染性腹膜炎病毒AH1905株N基因的生物信息学分析及原核表达

王元红^1,2, 邢雪^1,2, 李传峰², 朱杰², 王勇^1,*, 刘光清^2,*

1.安徽农业大学动物科技学院,安徽合肥 230036;
2.中国农业科学院上海兽医研究所,上海 200241

收稿日期:2019-08-05 出版日期:2020-03-25 发布日期:2020-04-03
通讯作者: *刘光清,E-mail:liugq@shvri.ac.cn;王勇,E-mail:wangyong119@ahau.edu.cn
作者简介:王元红(1995—),女,安徽芜湖人,硕士研究生,主要从事宠物病毒分子生物学研究。E-mail: 806576709@qq.com
基金资助:
国家重点研发计划(2016YFD0500108,2016YFD0501003); 上海市科技兴农创新项目(沪农科创字〔2019〕第3-3号); 中央级公益性科研院所基本科研业务费专项资金(2019JB06); 安徽农业大学研究生创新基金(2019ysj-36)

Bioinformatics analysis and prokaryotic expression of FIPV AH1905 strain N gene

WANG Yuanhong^1,2, XING Xue^1,2, LI Chuanfeng², ZHU Jie², WANG Yong^1,*, LIU Guangqing^2,*

1.College of Animal Science and Technology, Anhui Agricultural University, Hefei 230036, China;
2.Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China

Received:2019-08-05 Online:2020-03-25 Published:2020-04-03

摘要/Abstract

摘要： 为了解猫传染性腹膜炎病毒(feline infectious peritonitis virus, FIPV)在中国的流行与遗传变异情况,对新分离的FIPV AH1905株的N基因进行了克隆和序列比对分析,并利用生物信息学软件对N基因编码蛋白的二级结构进行了预测。最后,将N基因克隆至原核表达载体pGEX-4T-1,在原核细胞中进行表达,并制备鼠源多克隆抗体。测序结果表明,FIPV AH1905株的N基因全长1 134 bp,编码377个氨基酸。序列比对分析结果显示,FIPV AH1905株与报道的FIPV参考毒株的核苷酸同源性为90.2%~92.4%,氨基酸同源性为91.8%~93.9%,与国内其他分离株位于同一进化分支,同属于FIPV基因Ⅰ型。对N蛋白二级结构预测结果显示,该蛋白具有高度亲水性,其二级结构主要由α螺旋(h)(14.06%)、延伸链(e)(15.12%)、β转角(t)(3.71%)和无规则卷曲(c)(67.11%)组成,无信号肽区域和跨膜结构域,存在48个潜在的磷酸化位点,含有6个潜在的B细胞抗原表位、2个CTL表位和2个Th表位。Western blot检测结果证明,N蛋白在大肠埃希菌细胞中主要以包涵体形式大量表达,相对分子质量约为68 ku,具有良好的反应原性。以上结果可为进一步开展FIPV的流行病学和分子生物学研究奠定基础。

关键词: 猫传染性腹膜炎病毒, N基因, 原核表达, 生物信息学

Abstract: To understand the molecular epidemiological and genetic variation characteristics of feline infectious peritonitis virus (FIPV) in China, the N gene of FIPV AH1905 strain was cloned by PCR, and the bioinformatics softwares were used to predict the N protein. Then, N gene was cloned into a prokaryotic vector pGEX-4T-1 and was successfully expressed. The expressed protein was purified, and identified by SDS-PAGE and Western blot. The results showed that the N gene of FIPV, 1 134 bp, encodes 377 amino acids. The molecular bioinformatics analysis showed that the nucleotide homology and the amino acid homology between FIPV AH1905 strain and the reported FIPV reference strains were 90.2%-92.4% and 91.8%-93.9%, respectively. The phylogenetic analysis showed that FIPV AH1905 belongs to the FIPV gene type Ⅰ, the same as other isolates in China. The prediction of the secondary structure of the N protein showed that the protein was a hydrophilic stable protein with 14.06% α-helix (h), 15.12% extended chain (e), 3.71% β turn (t) and 67.11% random spiral (c). There were no signal peptide region and transmembrane domain. It may have 48 phosphorylation sites. Moreover, there may exist six B cell epitopes, two CTL epitopes and two Th epitopes. The molecular weight of expressed protein is approximately 68 ku, mainly expressed in inclusion body form with good reactogenicity. In conclusion, the present study has successfully expressed the N protein of FIPV, and prepared the multi-antiserum, which laid the foundation for further research on epidemiology and molecular biology of FIPV.

Key words: feline infectious peritonitis virus (FIPV), N gene, prokaryotic expression, bioinformatics

中图分类号:

S855.3

王元红, 邢雪, 李传峰, 朱杰, 王勇, 刘光清. 猫传染性腹膜炎病毒AH1905株N基因的生物信息学分析及原核表达[J]. 浙江农业学报, 2020, 32(3): 406-414.

WANG Yuanhong, XING Xue, LI Chuanfeng, ZHU Jie, WANG Yong, LIU Guangqing. Bioinformatics analysis and prokaryotic expression of FIPV AH1905 strain N gene[J]. , 2020, 32(3): 406-414.

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猫传染性腹膜炎病毒AH1905株N基因的生物信息学分析及原核表达

Bioinformatics analysis and prokaryotic expression of FIPV AH1905 strain N gene

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