摘要
为了构建犬瘟热病毒F基因真核表达载体,利用RT-PCR方法扩增出水貂源犬瘟热病毒分离株的F基因,将其克隆至pMD-18T载体上,用BamHⅠ和KpnⅠ进行双酶切,回收目的基因。将目的基因亚克隆至pcD-NA3.1(+)中,获得真核重组质粒pcDNA3.1-CDVF。通过脂质体介导法将pcDNA3.1-CDVF转染至BHK-21细胞中,并利用间接免疫荧光试验和RT-PCR法检测pcDNA3.1-CDVF在BHK-21细胞中的表达情况。结果表明,真核重组表达质粒pcDNA3.1-CDVF构建成功,F基因可在BHK-21细胞中表达。
In order to construct eukaryotic expression plasmid for F gene of canine distemper virus,the F gene of CDV strain isolated from mink was amplified by reverse transcription polymerase chain reaction （RT-PCR）. The amplified fragments were cloned into pMD-18T simple vector. Objective gene was obtained by digesting the cloning plasmid with BamHⅠ and Kpn Ⅰ ,and the eukaryon recombinant expression plas- mids was constructed by re-cloning the objective gene into pcDNA3.1 （＋） vector. Eukaryon recombinant expression plasmid pcDNA3.1-CDVF was transfected into BHK-21 cell by using lipoplast mediate method and it was proved with indirect fluorescent antibody technique and RT-PCR detection that the objective gene was successfully expressed in BHK-21 cell. The results of the study provided fundamental information for the further study on the prevention of mink CD and new generation vaccine.
出处
《西北农林科技大学学报（自然科学版）》
CSCD
北大核心
2007年第8期24-28,共5页
Journal of Northwest A&F University(Natural Science Edition)
关键词
水貂
犬瘟热病毒
F基因
真核表达
mink
canine distemper virus
F gene
eukaryotic expression
