摘要
采用RT-PCR技术从猪外周血单核细胞(PBMC)中获得猪白细胞抗原基因座P1、P14等位基因的胞外区,并在其3′端拼接上依赖BirA酶的可生物素化序列(BSP),构建了pET-P1-BSP和pET-P14-BSP高效表达载体并进行诱导表达和纯化,用SDS-PAGE和Western blotting法对表达产物进行鉴定。结果显示,成功构建了融合表达载体pET-P1-BSP和pET-P14-BSP,表达产物以包涵体的形式存在,获得高纯度的P1-BSP和P14-BSP蛋白,为进一步利用亲和素在体外构建SLA-I类分子肽四聚体奠定了基础。
Expression vector containing fusion protein of P1-BSP and P14-BSP was constructed to obtain purified protein for the assembling of swine leukocyte antigen class Ⅰ （SLA- Ⅰ ）,major histocompatibility complex protein class Ⅰ -peptide tetramer. The extracellular domains of two loci（P1 and P14）of SLA- Ⅰ were cloned and sequenced from swine PBMC by RT-PCR and BirA substrate peptide（BSP） sequence was added to the 3＇end of Pl and P14 genes. Then they were subcloned into pET-28a（ ＋ ）vector and expressed in BL21 （DE3）. The expressed fusion proteins P1-BSP and P14-BSP were purified and identified using SDS-PAGE and Western Blotting. Results showed the P1-BSP and P14-BSP fusion protein were successfully expressed and purified. These lay the foundation for the construction of SLA- Ⅰ complex to explore the cell-mediated immunity of the swine.
出处
《中国兽医学报》
CAS
CSCD
北大核心
2008年第4期456-460,共5页
Chinese Journal of Veterinary Science
基金
国家“863”计划资助项目(BA2006AA10A203)
黑龙江省自然科学基金资助项目(ZJN-0602-01)
