野桑蚕GST-Omega1基因在草地贪夜蛾Sf9细胞中的表达  

Expression of an omega class glutathione S-transferase gene from Bombyx mandarina in the Sf9 cell

作　　者：马晓英[1] 李兵[1] 贡成良[1] 沈卫德[1] 

机构地区：[1]苏州大学生命科学学院

出　　处：《昆虫学报》2007年第10期996-1001,共6页Acta Entomologica Sinica

基　　金：国家重点基础研究发展计划"973"项目(2005CB121005);国家自然科学基金项目(30471309)

摘　　要：谷胱甘肽S-转移酶(glutathione S-transferases,GSTs)是昆虫的重要解毒酶之一。为了研究野桑蚕Bombyxmandarina中谷胱甘肽S-转移酶在真核表达系统中的表达情况。本研究通过RT-PCR从野桑蚕中肠中获得GST-Omega1基因的cDNA序列,该基因的开放读码框为771bp,编码256个氨基酸。对推导的氨基酸序列用NCBI的蛋白质Conserved Domains工具进行在线分析,结果显示GST-Omega1的氨基酸序列中具有Cys38和8个GSH结合位点的Omega类基因保守序列。对所获得的基因克隆进表达载体pFastBacHT b中获得pFast-GST-Omega1,将其转化DH10Bac感受态细胞,获得Bac-GST-Omega1重组病毒DNA,用脂质体法转染草地贪夜蛾Sf9细胞,获得重组病毒。对表达产物经SDS-PAGE和Western blotting分析,能检测到一条分子量约为33kD的特异性条带,与推导的融合蛋白大小相符,该目的蛋白的表达量占总蛋白的14.4%。目的蛋白经His.Bind树脂纯化,用Lineweaver-Burk作图法测定其K和V,结果显示其K为2.81μmol/L,V为2.70μmol/(mg.min)。Glutathione-S-transferases （GSTs） were a class of the important xenobiotics-metabolizing enzymes in insects. In this research, a 771 bp GST-Omegal gene （encoded 256 amino acids） was cloned by RT-PCR from the midgut of Bombyx mandarina for exploring the expression profiling of enzyme GSTs in eukaryotic expression system. Analysis by Conserved Domains online tool of NCBI website indicated that the deduced amino acid sequence was comprised of the omega family conserved sequences with one Cys residue and eight GSH binding sites. GST-Omegal was then cloned into the pFastBacHT b vector, and the recombinant vector was transformed into DH10Bac competent cells to obtain the bacmid DNA. Spodoptera frugiperda Sf9 cells were transfected with the complexes of bacmid DNA and lipofectin to get recombinant baculovirus. The SDS- PAGE and Western blotting results of the expression of recombinant protein showed that a specific band was detected around 33 kD, consistent with the expected size of the fusion protein. The purified target protein by His-Bind resin could catalyze the universal GST substrate CDNB, and exhibited enzymatic activity Km 2.81 μmol/L and Vmax 2.70 μmol/（mg·min）.

关 键 词：野桑蚕 谷胱甘肽S-转移酶基因 克隆 表达 杆状病毒表达系统 SF9细胞 

分 类 号：Q966[生物学—昆虫学]