5株猪繁殖与呼吸综合征病毒ORF7全长基因的克隆及序列分析  

Cloning and phylogenetic analysis of the ORF7 full length gene of four strains of Porcine reproductive and respiration syndrome

作　　者：贾怀杰[1,2] 景志忠[1,2] 房永祥[2] 王晓霞[2] 陈国华[2] 李克斌[1,2] 窦永喜[2] 瞿惠玲[1,2,3] 杨孝朴[1] 

机构地区：[1]甘肃农业大学动物医学院,甘肃兰州730070 [2]中国农业科学院兰州兽医研究所,家畜疫病病原生物学国家重点实验室,农业部兽医公共卫生重点实验室,甘肃兰州730046 [3]甘肃省兽医局,甘肃兰州730030

出　　处：《中国病原生物学杂志》2009年第8期561-566,共6页Journal of Pathogen Biology

基　　金：国家自然科学基金项目(No.30871884);国家支撑计划项目(No.2006BAD31B03);甘肃省支撑计划项目(No.0804NKCA076)

摘　　要：目的通过测定5株猪繁殖与呼吸综合征病毒（PRRSV）ORF7基因序列,结合GenBank登录的国内外PRRSV分离株核苷酸序列,分析PRRSV核衣壳蛋白（N蛋白）的遗传变异情况。方法根据GenBank公布的PRRSV ATCCVR-2332株全基因组核苷酸序列,设计合成1对特异性引物,用RT-PCR方法扩增出3株PRRSV甘肃流行株（命名为GSZY、GSBY、GSZY株）、1株陕西流行株（命名为Shanxi株）和1株PRRSV江西分离株（命名为Jingxi株）的ORF7全长基因,克隆到pGEM-T Easy载体,然后分别测定插入的核苷酸序列。应用DNAStar序列分析软件对所测ORF7基因核苷酸序列及其推导的氨基酸序列与不同来源的13个PRRSV毒株进行同源性比较。结果5株PRRSV ORF7基因与以ATCC VR-2332为代表的早期引发PRRS的美洲型毒株的核苷酸序列间的同源性为91.9%～94.3%,与国内其他PRRSV分离株的同源性为98.7%～99.7%,与欧洲型代表株LV的同源性为57.7%～58.2%。结论美洲型PRRSV N蛋白氨基酸发生了变异,但国内毒株相对保守。Objective The objective of this study was to analyze the genetic variation of the nucleoeapsid protein （N protein） in Porcine Reproductive and Respiratory Syndrome Virus （PRRSV） in China by cloning and sequencing the ORF7 gene of 5 PPRSV strains and comparing it to other PRRSV epidemic strains in GenBank. Methods One pair of primers was designed according to the published full genome sequence of the ATCC VR-2332 strain in GenBank; the ORF7 full length gene of three Gansu epidemic strains （named GSZY, GSBY, and GSLZ strains）, one Shanxi epidemic strain （named the Shanxi strain）, and one Jiangxi epidemic strain （named the Jiangxi strain） were amplified by RT-PCR. The PCR products were cloned into a pGEM-T Easy vector, and recombinant vectors were constructed and then sequenced. Multiple sequences of ORF7 gene nucleotides and amino acids were compared with those of 13 PRRSV strains and analyzed using DNA Star software. Results Results indicated that the ORF7 gene shared 91.9% -94. 3% homology of nucleotides with an American virus （ATCC VR-2332 strain）, which caused the variation of PRRS in the early stages, while shared 98.7%-99.7% homology with other epidemic strains in China, and 57.7%-58.2% homology with a European strain （LV strain）. Conclusion Results showed that there is variation in the N protein （amino） acid （sequence） of the American strain but that it is relatively conserved among strains in China.

关 键 词：PRRSV 地方毒株 ORF7 基因克隆 变异分析 

分 类 号：S852.65[农业科学—基础兽医学]