摘要
根据GenBank中登录的犬瘟热病毒f基因序列设计一对引物,利用RT-PCR方法扩增出水貂源犬瘟热病毒分离株的f基因,将其插入到克隆载体pMD18-T中,经EcoRⅠ和XbaⅠ双酶切鉴定后纯化回收,与经同样的酶酶切消化后并纯化回收的载体pPICZaA连接并转化E.coliDH5α,在含Zeocin的低盐LB固体培养基上筛选阳性转化子,阳性克隆菌经PCR法鉴定、酶切分析和测序鉴定表明目的基因插入位置、方向和阅读框正确。证明pPICZaA-f酵母表达载体构建成功。
A pair of primers was designed based on the f gene sequence of canine distemper virus in Genbank, the f gene of the CDV strain isolated from mink was amplified by reverse transcription polymerase chain reaction （RT-PCR）. The amplified fragment was cloned into pMD18-T simple vector for sequencing, then objective gene was obtained by digesting the cloning plasmid with Eco R I and Xba I , and ligated to pPICZaA vector at the same sites to construct the eukaryon expression plasmid pPICZaA-f. Host strain E. coli DHSctwas transformated by pPlCZaA-f vector, positive recombinant transformant was selected by low salt LB medimn with Zeocin and identifieated by PCR, restriction analysis by EcoR I/Xba I and DNA sequencing. The results showed that intact pPICZaA-f recombinant expression vector were constructed successfully, and could be used for further expression in Piehia pastoris cells.
出处
《经济动物学报》
CAS
2008年第4期209-213,共5页
Journal of Economic Animal
基金
国家863计划(2007AA10Z443)
关键词
水貂
犬瘟热病毒
F基因
酵母表达载体
mink
canine distemper vires
f gene
yeast expression vector
