摘要
利用杆状病毒表达系统对H 5亚型禽流感病毒HA基因在sf9细胞中的表达进行了研究。首先将pUCm-T-H 5HA质粒用B amHⅠ及N otⅠ酶切,获得HA基因片段,同时杆状病毒转座载体质粒pF astB acⅠ也用B amHⅠ及N otⅠ酶切,然后用T4DNA连接酶连接,构建了重组质粒pF astB ac-H 5HA。再将该重组质粒转化DH 10 B ac感受态细菌,在体内进行重组,并经三重抗性与蓝白斑筛选,得到杆状病毒重组质粒B acm id-H 5HA。将B acm id-H 5HA转染sf9细胞,获得重组杆状病毒,经SDS-PAGE和W estern b lotting检测表明,HA蛋白在重组杆状病毒中获得表达。
According to the HA gene of H5 AIV from pUCm-T-HA,the objective fragment （1 707 bp） was obtained. Then the HA gene was cloned into baculovirus expression vectors pFastBac I ,and the recombinant plasmid of pFastBac-H5HA was constructed. The plasmid were transformed to DH10 Bac competent cell ,and screened by three antibiotics and blue-white patch, the white colony was proved to be the recombinant Bacmid-H5HA. And then the Bacmid-H5HA was transfected into the sf9 insect cells,by three passaging the recombinant baculovirus was obtained. Finally,the expressed product were detected by SDSPAGE and Western blotting. The results showed that the polymerase （HA protein） was high-efficiently expressed in the recombinant baculovirus.
出处
《中国兽医学报》
CAS
CSCD
北大核心
2006年第4期387-389,共3页
Chinese Journal of Veterinary Science
基金
国家"十五"科技攻关计划资助项目(2004BA519A12)
关键词
禽流感病毒
HA基因
杆状病毒
表达
avian influenza virus
HA gene
baculovirus
expression
