意大利蜜蜂蜂毒磷脂酶A_2基因在杆状病毒-昆虫细胞系统中的表达  

Expression of phospholipase A_2 gene from the venom of Apis mellifera in the baculovirus-insect cell system

作　　者：沈立荣[1] 邢丽苹[1] 张传溪[1] 程家安[1] 

机构地区：[1]浙江大学应用昆虫学研究所,杭州310029

出　　处：《昆虫学报》2006年第3期367-372,共6页Acta Entomologica Sinica

基　　金：国家自然科学基金项目(30271008)

摘　　要：利用BactoBac系统将意大利蜜蜂蜂毒磷脂酶A2(AmPLA2)基因cDNA克隆至转移载体pFastBacHTa中,得到pBacHT-AmPLA2,再将其转化入含穿梭载体Bacmid的受体大肠杆菌DH10Bac中,通过转座作用,得到含AmPLA2基因的重组病毒rBacmid-AmPLA2的DNA。提取其基因组DNA,用脂质体介导转染粉纹夜蛾细胞Tn-5B1-4,得到重组病毒rACV-Bac-AmPLA2。用此重组病毒感染Tn-5B1-4细胞,在细胞中表达AmPLA2。SDS-PAGE电泳结果显示,与6×HisTag融合表达的产物蛋白分子量约为18kD左右,表达量约占细胞总蛋白的5·35%。Westernblot印迹显示,融合表达产物能与意大利蜜蜂蜂毒AmPLA2抗血清发生免疫反应。生物活性测定显示,含表达产物的细胞蛋白粗提物对底物蛋黄的酶活力约为6·13μmol·min-1·mg-1。The cDNA encoding phospholipase A2 of Apis mellifera （AmPLA2 ） was cloned into a transfer vector pFastBacHTa to form the recombinant donor plasmid pBacHT-AmPLA2. The recombinant donor plasmid was then transformed into Escherichia coli DHIOBac. By transposition, AmPLA2 gene was integrated into Bacmid, and a recombinant shuttle vector, rBacmid-AmPLA2 was constructed. The cultured Trichoplusia ni Tn-5B1-4 cells, mediated with Lipofectin, were transfected with the rBacmid-AmPLA2 DNA, and then the recombinant baculovirus, rACV-Bac-AmPLA2 was obtained. The recombinant virus was further used to infect the Tn-5B1-4 cells to express the target protein. SDS-PAGE analysis of the infected cellular proteins showed that the size of the expression product of AmPLA2 fused with 6 × His-tag at its N-terminal was about 18 kD, and the expressed protein accumulated up to about 5.35% of the total cellular proteins. Western blot analysis using antiAmPLA2 polyclonal serum confirmed the expressed protein was a fusion protein of AmPLA2. The protein extracts of AmPLA2 showed an enzymatic activity of about 6.13 μmol· min^-1· mg^-1 for hydrolyzing egg yolk substrate.

关 键 词：意大利蜜蜂 蜂毒 磷脂酶A2基因 杆状病毒-昆虫细胞系统 表达 

分 类 号：Q966[生物学—昆虫学]