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.
出处
《昆虫学报》
CAS
CSCD
北大核心
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)
