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.
出处
《中国兽医学报》
CAS
CSCD
北大核心
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 )
