昆虫细胞中表达eGFP基因的重组杆状病毒的构建  

Construction of an Enhanced Green Fluorescent (eGFP) Tagged Dinsect-baculovirus Transference Vector

作　　者：唐晓艳[1] 高冬妮[1] 李梅[1] 葛菁萍[1] 平文祥[1] 楼庄伟[1] 

机构地区：[1]微生物黑龙江省高校重点实验室/黑龙江大学生命科学学院,哈尔滨150080

出　　处：《中国农学通报》2013年第8期26-30,共5页Chinese Agricultural Science Bulletin

基　　金：黑龙江大学高层次人才(创新团队)支持计划"功能微生物及其产物应用"(Hdtd2010-17)

摘　　要：为构建表达增强型绿色荧光蛋白(eGFP)融合蛋白的昆虫杆状病毒转移载体。利用Sf-9细胞表达eGFP,用PCR法从重组Bacmid和pEGFP-C3质粒中扩增gp64SP、eGFP和VSVGED片段,利用一步法SOE-PCR将3个片段连接在一起。用XbaI和HindIII酶切融合片段与pFastBacPH,连接获得阳性质粒命名为pFB/LM-PH。转化E.coliDH10Bac,通过转座子Tn7的介导,获得重组杆状病毒Bv-LM-PH,感染Sf9昆虫细胞。结果表明:通过倒置荧光显微镜观察细胞形态及荧光表达情况;结果证实:感染的Sf9细胞可表达外源基因eGFP。重组pFB/LM-PH载体具备表达eGFP的能力,为分子生物学研究提供了一种新型可表达eGFP融合蛋白的昆虫杆状病毒表达载体。To construct an enhanced Green Fluorescent Protein （eGFP） tagged insect-baculovirus transference system and expressed eGFP by Sf-9 cells.After gp64SP and TM/CTD gene had been amplified from bacmid by PCR,and eGFP gene had been amplified from pEGFP-C3 plasmid by PCR,the three fragments were connected together by SOE-PCR.The recombinant gene digested by Xba I/Hind Ⅲ,it was recombined into baculovirus transference vector pFastBac[PH] to create a novel pFB/LM-PH vector.E.coli DH10Bac was transformed with the pFB/LM-PH vector,a recombinant baculovirus Bv-LM-PH,was obtained with the action of Tn7 transposon.The insect Sf-9cells were infected with the recombinant baculovirus Bv-LM-PH and expressed eGFP.Meanwhile,the Sf 9cells infected by the recombinant baculovirus were observed under inverted fluorescence microscope.The eGFP expression in infected Sf 9cells was proved by inverted fluorescence microscope.The bright green fluorescence was demonstrated in plasma and nuclei of infected Sf-9cells.The novel pFB/LM-PH baculovirus expression system could expressed eGFP efficiently and could became a useful eGFP tagged expression system for detection of the interaction of proteins.

关 键 词：昆虫杆状病毒表达系统 增强型绿色荧光蛋白 昆虫细胞 

分 类 号：S852.65[农业科学—基础兽医学]