鹦鹉幼雏病毒结构蛋白VP1基因的克隆及原核表达

Cloning and Expression in E.coli of Structure Protein VP1 Gene of Budgerigar Fledgling Disease Virus

采用湖北省云梦县患鹦鹉幼雏病死亡的虎皮鹦鹉体内分离得到的鹦鹉幼雏病毒 (budgerigarfledglingdiseasevirus,BFDV) ,应用PCR方法获得BFDV主要结构蛋白基因 (VP1) ,克隆到 pMD18 T载体 ,构建重组质粒 pMD18T VP1并进行测序 ,结果显示 ,结构蛋白基因 (VP1)与BFDV欧美分离株的同源性为 96 %～ 99% ,表明VP1是BFDV保守基因 ;将VP1亚克隆到原核表达载体pET32 (+)b ,构建重组质粒 pET32 (+)b VP1,转化菌株BL2 1(DE3) ,诱导表达 ,经SDS PAGE和Western blot分析 ,克隆在his tag下游的结构蛋白基因获得了高效融合表达 ,表达的融合蛋白分子量约为 5 5kD。为建立快速、灵敏的鹦鹉幼雏病毒血清学检测方法以及研制基因工程疫苗提供了科学依据。

The major structure protein gene (VP1) of Budgerigar fledgling disea se virus (BFDV) was isolated from BFDV genome by PCR. The gene was cloned into p MD18-T vector, the recombinant plasmid pMD18T-VP1 was sequenced and compared w ith other BFDV isolates. The result showed that the homologies between the clone d VP1 and BFDV isolated from America and Europe varied from 95% to 99%. So, the VP1 was highly conservative sequence in BFDV genome. The VP1 was subclon ed into prokaryotic expressing vector pET32, the recombinant plasmid named pET32 (+)b-VP1 was constructed. The pET32(+)b-VP1 was used to transform into E.col i BL21. The results of SDS-PAGE and Western-blot showed that the major struc ture protein gene cloned in the downstream of His-Tag was expressed in a high l evel and the molecular weight of the recombinant fusion protein was about 55 kD. This study lay a basic foundation on the development of the diagnosis methods i n serology and vaccines for BFDV.