人脂联素球状结构域基因在昆虫杆状病毒表达系统中的表达  

Expression of Human gAd Gene in Baculovirus-Insect Expression System

作　　者：安薇[1] 徐霞[1] 

机构地区：[1]广州医学院医学检验系,广州510182

出　　处：《热带医学杂志》2010年第4期372-376,F0002,共6页Journal of Tropical Medicine

基　　金：第八轮广东省高等学校重点扶持学科资助项目[粤教科(2007)26号]

摘　　要：目的利用杆状病毒表达系统表达人脂联素球状结构域(gAd)基因。方法以人基因组为模板,PCR法扩增人gAd基因,将gAd基因与供体质粒pFastBacHTB连接,转化含有穿梭载体Bacmid的大肠杆菌DH10Bac。筛选转座成功的重组穿梭载体Bacmid-gAd,通过脂质体介导,转染昆虫细胞Sf9,经SDS-PAGE、免疫印迹法检测表达产物。结果重组杆状病毒感染的Sf9细胞形态变化明显,SDS-PAGE电泳结果显示,Bacmid-gAd组和对照组相比,在相对分子质量15000～25000之间多出一清晰的蛋白条带,免疫印迹法证实该条带能与相应的抗His标签抗体结合。结论人gAd基因成功在真核细胞中表达,为后续的实验研究奠定了基础。Objective To express the human globular domain of adiponectin （gAd） gene with bac-to-bac baculovirus expression system. Methods Human gad gene was amplified by PCR method using human genomic DNA as template,and then inserted to donor plasmid of pFastBacHTB and transposed into DH10Bac containing a bacmid shuttle vector.Transposons containing Bacmid-gAd were selected and then transfected Sf9 cells in the mediation of liposome.The recombinant protein was detected by SDS-PAGE and western-blot. Results Sf9 cells infected by the recombinant baculovirns was observed for obvious morphological changes. SDS-PAGE electrophoresis results showed that an additional protein band with a molecular weight between 15 000 to 25 000 was observed in Bacmid-gAd group compared to control group. Western-blot proved that this protein can bind with the anti-His antibody. Conclusion Human gad gene was successfully expressed in Sf9 cells, which lay the foundation to the follow-up testing.

关 键 词：脂联素球状结构域 杆状病毒 昆虫细胞SF9 

分 类 号：Q78[生物学—分子生物学]