猪繁殖与呼吸综合症病毒BJ-4株生长cDNA的构建.pdfVIP

业生物技术学报 Journal of Agricultural Biotechnology 2005,13 (1): 61~65 ·研究论文· 猪繁殖与呼吸综合征病毒BJ-4 株全长cDNA 的构建* 陈小云 杨汉春** 郭 鑫 ( 中国农业大学农业部预防兽医学重点开放实验室，北京 100094) 摘要: 依据猪繁殖与呼吸综合征病毒 (PRRSV)BJ-4 株全基因组序列，分6 段设计引物A 1F/A 1R、A2F/A2R 、B 1F/B 1R、 B2F/B2R 、C1F/C1R 和D 1F/D 1R，应用RT-PCR 技术扩增出其全基因组cDNA 片段。将各cDNA 重叠片段 、 、 和 分别克隆入pGEM-T Easy 载体后，依次连接并克隆到低拷贝质粒载体pWSK29 ，获得该毒株全长cDNA 克隆pWSK 。 为进一步研究该毒株的感染性cDNA 克隆和深入研究PRRSV 的分子致病机理以及发展安全有效的活病毒疫苗奠定了基础。 关键词: 猪繁殖与呼吸综合征病毒; 全长cDNA; 分子克隆和构建 Construction of Full-length cDNA of BJ-4 Strain CHEN Xiao-Yun YANG Han-Chun** GUO Xin （ ） Six sets of primers were designed according to the full-length genomic sequence of porcine reproductive and respiratory syndrome virus (PRRSV) BJ-4 strain. Utilizing the RT-PCR, 6 overlapping cDNA fragments covering the full genome were amplified in the presence of primer pairs A 1F/A 1R, A2F/A2R, B 1F/B1R, B2F/B2R, C 1F/C 1R and D 1F/D 1R . These cDNA overlapping frag- ments , , , , and were cloned into pGEM-T Easy Vector . Then the full-length cDNA clone pWSK of PRRSV BJ-4 strain were obtained when the 6 fragments were ligated in order and cloned into low-copy-number vector pWSK 29. Successful construction of full-length cDNA provided fundamental materials for further study on the infectious cDNA clone of BJ-4 strain and the molecular pathogenisis of PRRSV, as well as for the development of a safe and effective live vaccine for use in pigs.