LA-PCR快速克隆传染性法氏囊病病毒基因组A节段全长cDNA方法的建立  

Construction of Full-length cDNA of Infectious Bursal Disease Virus Genomic Segment A by Long-accurate PCR

作　　者：李建荣[1] 宋厚辉[1] 于涟[1] 黄耀伟[1] 谢荣辉[1] 周继勇[1] 郑筱祥[2] 

机构地区：[1]浙江大学动物预防医学研究所,浙江杭州310029 [2]浙江大学生物医学工程研究所,浙江杭州310027

出　　处：《中国兽医学报》2002年第5期438-441,共4页Chinese Journal of Veterinary Science

基　　金：国家"8 6 3"计划资助项目 ( 10 1-j99-0 2 )

摘　　要：从浙江省某鸡场暴发疑似传染性法氏囊病 (IBD)的法氏囊病料中分离到 1株致死率高达 70 %的强毒株 (IBDV-ZJ2 0 0 0 )。通过筛选 ,采用效果最理想的蛋白酶 K法从法氏囊中提取病毒基因组 RNA,经 L i Cl纯化后 ,优化各种反应参数和条件 ,建立了 L ong- accurate PCR(L A- PCR)一步直接扩增 IBDV A节段全长 c DNA的方法 ,得到一约 3.2 6 kb的片段。L A- PCR法能快速从病鸡法氏囊和细胞适应毒中扩增 A节段全长 c DNA,为研究各 IBDV毒株 A节段的结构和功能打下了基础。An improved methods of reverse transcription, long-accurate polymerase chain reaction (LA-PCR) amplification, and cloning of full-length cDNA of segment A of infectious bursal disease virus were developed. After identification of a very virulent strain of infectious bursal disease virus(IBDV-ZJ2000) from Zhejiang province, the double stranded genomic RNA was extracted from infected bursae by the optimal proteinase K digestion based approach,and purified by LiCl, followed by the LA-PCR in a single step resulted in the synthesis of 3.26 kb of segment A. The amplified cDNA fragments were directly cloned into pGEM-T Easy Vector by T-A clone method. The results of PCR identification and analysis of restriction enzymes digestion indicated that the full-length cDNA of segment A had been successfully cloned. LA-PCR was specific, simple, convenient to amplify IBDV segment A either from sick bursae or chicken embryo fibroblast(CEF) adapted virus, and will greatly enhance the availability to research the structure and function of segment A.

关 键 词：家畜病毒学 LA-PCR快速克隆 传染性法氏囊病 病毒 基因组 A节段 CDNA 

分 类 号：S852.65[农业科学—基础兽医学] Q78[农业科学—兽医学]