摘要
根据已发表的鹅圆环病毒GoCV-yk1株的序列,设计引物扩增出全长核衣壳蛋白基因(Cap),用BamHⅠ和EcoRⅠ双酶切后插入到原核表达载体pET-28a中,转化大肠杆菌BL21(DE3)进行融合表达,经SDS-PAGE电泳检测未见到目的蛋白条带。应用生物信息学分析确定Cap蛋白的核定位信号区域,新设计一对引物扩增出不包含核定位信号序列编码区的核衣壳蛋白基因,将截短Cap基因克隆入pET-28a进行融合表达,经SDS-PAGE电泳和Western blot检测,截短Cap蛋白获得了高效表达,融合蛋白的分子量约为27.6ku,表达产物以包涵体形式存在,其表达量可以达菌体总蛋白的18.3%。截短Cap蛋白的高效表达为鹅圆环病毒ELISA检测方法的建立和进一步开展GoCV致病性及其免疫机制的探索奠定了基础。
The full-length Cap gene was amplified with a pair of primers designed based on the published goose circovirus （GoCV-ykl） sequence. The amplified DNA digested with BamH I and EcoR I was inserted into pET-28a vector. However, the Cap gene failed to expressed in BL21（DE3） analyzed by SDS-PAGE. Therefore, a truncated Cap gene without the coding region of nuclear localization signal was amplified with a new pair of primers. The gene was subcloned into pET-28a vector and transferred into BL21 （DE3） for expression. SDS-PAGE and Western blot analysis showed that the truncated Cap recombinant protein was about 27.6 ku and up to 18.3 % of the total bacteria proteins, mainly in form of inclusion body. The recombinant protein can be used to establish ELISA test which will be a tool to explore the mechanism of GoCV pathogenicity and host immunoresponse.
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2008年第7期505-509,共5页
Chinese Journal of Preventive Veterinary Medicine
基金
国家自然科学基金(30771608)
浙江省科技计划重点项目(2007C22051)
关键词
鹅
圆环病毒
核衣壳蛋白基因
核定位信号
原核表达
goose
circovirus
capsid protein gene
nuclear localization signal
prokaryotic expression
