牛病毒性腹泻病毒反转录及3'-末端克隆测序方法研究  

Reverse Transcription and 3'-Terminal Sequencing of the Bovine Viral Diarrhea Virus

作　　者：李庆超[1] 苗利光[1] 李海涛[1] 刘艳环[1] 张广雷[2] 庄风岐 

机构地区：[1]中国农业科学院特产研究所,吉林吉林132109 [2]江苏科技大学,江苏镇江212018 [3]吉林省桦甸市八道河子镇农村管理服务中心,吉林吉林132417

出　　处：《特产研究》2010年第2期12-15,共4页Special Wild Economic Animal and Plant Research

基　　金：中国农科院特产研究基金项目

摘　　要：牛病毒性腹泻病毒为黄病毒科瘟病毒属代表种,其基因组为单股正链RNA,无Poly(A)尾。传统的3'-末端快速扩增方法不适用于牛病毒性腹泻病毒的3'-末端克隆测序。本研究根据牛病毒性腹泻病毒基因组3'-末端共有特征碱基,设计回文锚定引物CGend。以BVDVJZ05-1毒株为研究对象,进行了反转录及3'-末端克隆测序。结果表明,CGend的反转录效果优于随机引物和一般特异性下游引物,并且扩增得到了JZ05-1的3'-末端完整序列。本研究为瘟病毒属病毒反转录和3'-末端克隆测序提供了新思路。Bovine viral diarrhea virus（BVDV）is a member of the genus Pestivirus.BVDV is containing a single positive-stranded RNA genome without poly（A）tail at 3＇-terminal,so that the rapid amplification of cDNA ends method can not use directly to clone the 3＇-terminal of BVDV genome.Consensus sequence CGn was found by multiple sequence alignment of 14 BVDV-2 entire genome sequences.Palindrome anchorage primer CGend was designed to reverse transcribing and amplifying the 3＇-terminal of the BVDV JZ05-1 genome.RT-PCR amplification products using primer CGend were much better compared with the random primer control and downstream gene specific primer.The full long 3＇-terminal sequence was cloned and sequenced.This simple and practicable method of reverse transcription and 3＇-terminal sequencing may be useful in research of other Pestivirus.

关 键 词：牛病毒性腹泻病毒 瘟病毒 反转录 3'-末端 

分 类 号：Q939.4[生物学—微生物学] Q753