猪繁殖与呼吸综合征病毒CH-1a株基因组全长cDNA克隆的构建  

Construction of a full length genomic cDNA clone of porcine reproductive and respiratory syndrome virus

作　　者：薛强[1] 周艳君[1] 刘光清[1] 仇华吉[1] 童光志[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室,黑龙江哈尔滨150001

出　　处：《中国预防兽医学报》2003年第6期401-407,共7页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家重点基础研究发展规划项目 (973)资助(G19990 1190 2 )

摘　　要：猪繁殖与呼吸综合征病毒 (PRRSV)嗜好在PAM和其他单核细胞上生长 ,如果把PRRSV作为一种病毒活载体 ,它就可以携带外源抗原高效进入猪体的免疫系统 ,更好地刺激机体产生免疫应答。本实验根据PRRSV基因组和克隆载体的限制性内切酶酶切位点 ,将PRRSV基因组设计为 8段进行了反转录和扩增 (RT_PCR) ,将得到的片段克隆到质粒pBluescriptIISK(_)或pMD 18T_vetcor中。在扩增 5’端时 ,运用 5’RACE获得了基因组 5’最末端序列 ,在亚克隆入pOK12前在 5’端上游引入了T7启动子序列。选取一个克隆在 3’端紧挨ORF7下游引入了PacⅠ位点 ,以期作为感染性分子克隆的分子标志。将扩增片段在pBuescriptIISK(_)中进行了部分连接后 ,亚克隆于低拷贝质粒pOK12中 ,在pOK12中进行了长片段的连接 ,并获得了基因组全长cDNA克隆。进行全基因测序后 ,在 3处发现有 6个核苷酸的缺失 ,运用插入突变修补丢失的碱基 ,最终获得了含有PRRSVCH_la株基因组全长cDNA的重组质粒pOKCH_laF和含有分子标志PcaⅠ的基因组全长cDNA克隆pOKCH_laPacIF。在本试验中构建的基因组全长cDNA克隆为进一步获得具有感染性的PRRSV分子克隆并研究该病毒基因组结构和功能奠定了基础。Since PRRSV specifically infects alveolar macrophages and other monocytes,which are involved in antigen presentation to the immune system,it is conceivable that attenuated mutants of the virus can be used as vectors for presenting antigens of other pathogens of pigs.In the study,eight cDNA fragments covering the PRRSV genome were amplified and cloned into pBluescript II SK(-)or pMD 18 T-vector.The 5'-utmost sequence was obtained by 5'RACE.A T7 promoter was introduced immediately upstream of 5'-end before the 5'-end fragment was cloned into the low-copy-number plasmid pOK12.A molecular marker,PacⅠ site,was introduced downstream of the ORF7 termination code.The amplified fragments were cloned in the pBluescript II SK(-) at first and then subcloned into pOK12.The recombinant plasmid pOKCH-la containing a full-length genomic cDNA of PRRSV,and pOKCH-laPacI with additional PacⅠ site,was obtained after several cycles of subcloning.But six-nucleotide deletions at three of four sites in the recombinant plasmids were found after sequencing.The deleted nucleotidess were inserted by mutagenesis and the repaired plasmids pOKCH-laPacIF were obtained.The construciton of a full-length genomic cDNA clone of PRRSV is a crucial step to obtain the infectious clone.Further study will be going to test infectivity of the cDNA clone and look insight the structure and function of PRRSV genome.

关 键 词：猪繁殖与呼吸综合征病毒(PRPSV) 基因组全长cDNA克隆 感染性分子克隆 

分 类 号：S852.65[农业科学—基础兽医学] Q784[农业科学—兽医学]