摘要
为获得贵州省猪繁殖与呼吸综合征病毒(PRRSV)流行毒株全基因组序列,根据GenBank上PRRSV VR-2332株设计合成8对特异性引物,对发生在贵州省安顺地区某养猪场PRRS疑似病例进行了PRRSV分离以及全基因组测序与分析。结果表明:组织病料悬液接种Marc-145细胞后,出现细胞皱缩、聚集成簇或脱落等细胞病变,并将分离毒株命名为PRRSV AS株;采用8对PRRSV引物对接种细胞RNA提取物进行RT-PCR扩增,均可得到与预期大小(1 057bp,20 16bp,1 771bp,1 902bp,2 263bp,2 569bp,2 734bp,3 778bp)相一致的特异性条带;对RT-PCR产物测序后进行比对和拼接,得到PRRSV AS株基因组全长为15 338bp,其中G+C含量为52.2%,与欧洲型LV株、美洲型VR-2332株、中国早期毒株CH-1a和新近变异毒株JXA1的核苷酸相似性分别为61.5%、89.1%、94.2%和98.1%。成功获得了贵州省安顺地区PRRSV流行毒株即AS株全基因组。
In order to obtain whole genome of the PRRSV epidemic strain in Guizhou,eight pairs of primers were designed based on the genome of PRRSV VR-2332strain in GenBank,by application of RNeasy Plus Mini Kit,the virus RNA were extracted from infected Marc-145cells which were vaccinated with samples by appropriate treatment of lungs and lymph nodes of swine infected by PRRSV,and were used for amplification by RT-PCR,then recovered DNA products and connected to pMD18-T,finally transformed into E.coli DH5a.The PCR products were sequenced and analyzed after the screening and identification of blue and white colony.The results showed as following:After the inoculation of treated specimen,cells shrunk,clustered,or exfoliated in Marc-145cells,the PRRSV was successfully isolated and named as PRRSV AS strain.Expected size of 1 057bp,2 016bp,1 771bp,1 902bp,2 263bp,2 569bp,2 734bp,and 3 778bp were amplified by 8pairs of PRRSV primers from RNA extracts of the inoculated cells.The whole genome of PRRSV AS strain was obtained after these PCR products were sequenced and aligned,with length of 15 338bp,and the content of G+C was 52.2%.The similarity of nucleotide sequence were 61.5%,89.1%,94.2%,and 98.1%,respectively with strains of European LV,American VR-2332,China CH-1a,and recently mutated JXA1.The above achievement indicated that the whole genome of PRRSV AS strain was successfully obtained in this study in Anshun,Guizhou.
出处
《贵州农业科学》
CAS
北大核心
2013年第10期127-131,共5页
Guizhou Agricultural Sciences
基金
贵州省重点实验室项目"贵州省动物疫病与兽医公共卫生重点实验室(培育)"[黔科合计Z字(2011)4008]
