摘要
为鉴定高致病性猪繁殖与呼吸综合征病毒(HP-PRRSV)HuN4株感染猪肺泡巨噬细胞(PAM)后,细胞内转录发生差异的基因,本研究采用抑制性消减杂交技术,以HP-PRRSV HuN4株感染后48h的PAM细胞mRNA为实验方(Tester),以未感染的PAM细胞为驱动方(Driver)进行消减杂交,得到了HP-PRRSV HuN4株感染后PAM内转录后发生上调的基因。反之,实验方与驱动方互换进行消减杂交,得到了HP-PRRSV HuN4株感染后PAM内转录后发生下调的基因。将2种杂交所得的差异转录基因经PCR扩增后分别克隆到T载体中并转化大肠杆菌感受态细胞,从而构建了正向(上调)和反向(下调)的差异cDNA文库。随机挑取文库中的多个克隆用PCR方法进行鉴定,结果表明差异文库中的cDNA具有较好的多样性。本研究为下一步的文库克隆的序列测定与差异基因的功能分析奠定了基础。
To investigate the gene transcription and expression related to virus infection, suppression subtractive hybridization technique was used to identify the up-regulated or down-regulated genes in the target cells （PAM） following PRRSV infection. In forward-subtraction, the cells infected by highly pathogenic porcine reproductive and respiratory syndrome virus （HP-PRRSV） HuN4 strain were taken as tester and non-infected as driver; In reverse-subtraction, the non-infected cells were taken as tester and infected as driver. The up-regulated and down-regulated genes were PCR amplified, ligated and transformed into E.coli cells to obtain differential transcription gene library. Nested PCR confirmed the high diversity of the library.
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2009年第1期37-41,共5页
Chinese Journal of Preventive Veterinary Medicine
基金
国家基础研究973基金项目(2005CB523200)
作者简介
肖燕（1981-），女，河南安阳人，硕士研究生，主要从事动物病毒分子生物学研究．
通讯作者：E-mail：gztong@hvri．ac．cn
