中国农业科学 ›› 2010, Vol. 43 ›› Issue (4): 828-834 .doi: 10.3864/j.issn.0578-1752.2010.04.021

• 兽医 • 上一篇    下一篇

猪瘟病毒3′非编码区的多态性及其二级结构分析

范运峰,赵启祖,赵耘,邹兴启,张仲秋,王琴,宁宜宝

  

  1. (中国兽医药品监察所)
  • 收稿日期:2009-04-09 修回日期:2009-05-22 出版日期:2010-02-20 发布日期:2010-02-20
  • 通讯作者: 范运峰

Heterogeneity and Secondary Structure Analysis of 3′ Untranslated Region in Attenuated Classical Swine Fever Viruses

FAN Yun-feng, ZHAO Qi-zu, ZHAO Yun, ZOU Xing-qi, ZHANG Zhong-qiu, WANG Qin, NING Yi-bao
  

  1. (中国兽医药品监察所)
  • Received:2009-04-09 Revised:2009-05-22 Online:2010-02-20 Published:2010-02-20

摘要:

【目的】分析CSFV 3′-UTR一级序列和二级结构,为研究其对病毒复制、增殖和毒力的关系奠定良好的基础。【方法】利用RT-PCR技术扩增CSFV Thiverval株、HCLV株和Shimen株的3′-UTR并测序,使用DNAstar、Sequencher和RNAstructure软件进行CSFV 3′-UTR基因组序列的比对分析和二级结构模拟。【结果】Thiverval株3′-UTR最长可达259个碱基,与母源株Alfort相比,含有32 nt的插入,最短只有233个碱基,含有6 nt的插入。HCLV株最长有244个碱基,含有17 nt的插入;最短有233个碱基,含有8 nt的插入。同时,疫苗株的插入片段导致3′-UTR空间构型和自由能发生改变,自由能随着插入片段的增加而升高。【结论】(1)CSFV 3′-UTR存在两个高度变异的区域。(2)CSFV疫苗株Thiverval株和HCLV株3′-UTR同一样品不同克隆株中插入片段呈现明显的不均一。(3)疫苗株的插入片段影响3′-UTR二级结构的稳定性,可能是导致疫苗株毒力减弱的原因之一。

关键词: 猪瘟病毒, 3′-UTR, 不均一性, 二级结构分析

Abstract:

【Objective】 The 3′-UTR of CSFV was sequenced and analyzed, for the understanding of the influence of 3′-UTR on replication, multiplication, and virulence of virus. 【Method】 The 3′-UTR of CSFV Thiverval, HCLV and Shimen strains were amplified and sequenced using RT-PCR method. The alignment was carried out using DNAstar and Sequencher software. The RNA secondary structures of different 3′-UTR were predicted using RNAstructure software. 【Result】 The longest 3′-UTR of Thiverval strain was 259 base pairs (bp) with a 32 nt insertion. The shortest 3′-UTR had only 233 bp with a 6 nt insertion. The longest 3′-UTR of HCLV strain was 244 bp with a 17 nt insertion and the shortest 3′-UTR was 235 bp with a 8 nt insertion. At the same time, to maintain the similar 3′-UTR structure, the free energy of vaccine strain is higher than that of virulent strain, and the free energy in 3′-UTR of vaccine strain is rising with length of insert. 【Conclusion】 The 3′-UTR of CSFV vaccine strains have two variable regions. The insertion of attenuated Thiverval and HCLV strains 3′-UTR represent distinct heterogeneity in different clones of the same sample. The insertion of vaccine strains 3′-UTR could affect the stability of the secondary structure, which might be a reason for the attenuated virulence.

Key words: classical swine fever virus, 3′-UTR, heterogeneity, RNA secondary structure