大鼠甘氨肽α-羟化单氧酶在昆虫细胞中的活性表达  

FUNCTIONAL EXPRESSION OF RAT PEPTIDYLGLYCINE α-HYDROXYLATING MONOOXYGENASE IN INSECT CELLS

作　　者：江智红[1] 黄荣[1] 杨宇虹[1] 吴祥甫[1] 李伯良[1] 王德宝[1] 

机构地区：[1]中国科学院上海生物化学研究所

出　　处：《病毒学报》1998年第3期246-252,共7页Chinese Journal of Virology

基　　金：国家"863"高技术研究发展计划资助

摘　　要：将编码大鼠甘氨肽α－羟化单氧酶（ＰＨＭ）ｃＤＮＡ基因，插入昆虫杆状病毒转移表达载体ｐＢａｃＰＡＫ８，构建成表达质粒ｐＢａｃＰＨＭ２，与修饰的银纹夜蛾核多角体病毒ＢａｃＰＡＫ６线性化ＤＮＡ共转染秋粘虫细胞Ｓｆ２１，通过同源重组，得到在核多角体蛋白基因启动子控制下的ＰＨＭ基因的重组病毒ＢａｃＰＨＭ。用ＢａｃＰＨＭ感染Ｓｆ２１细胞，无血清培养上清在７２小时后检测到酰胺化酶最高活力；用细胞免疫组化法和免疫印迹法检测表达产物，胞内及胞外培养上清液中均显示有约４１ｋＤ的ＰＨＭ表达产物，胞内、胞外产量分别约为５μｇ／１０５细胞数、１μｇ／ｍｌ培养基／１０５细胞数，且分泌至培养基中的酶活力远高于胞内。A recombinant plasmid transfer vector,pBacPHM2,was constructed by inserting peptidylglycine α-hydroxylating monooxygenase(PHM)cDNA into transfer vector pBacPAK8. The recombinant Autographa californica nuclear polyhedrosis virus BacPHM was generated by cotransfection of pBacPHM with the linear genomic DNA of modified AcNPV into Sf21 cells.The PHM activity reached to the peak 72 hours after Sf21 cells were infected with the recombinant virus.By immunohistochemical analysis,the 41kD product can be detected in the cell extract and in the culture medium with the yield of 5μg/10 5 cells and 1μg/ml medium/10 5 cells,respectively.The amidating activity assay showed that the active PHM secreted in the medium was much higher than that extracted from the cells.PHM expressed by insect cells can be directly used in amidation of Gly-terminated peptides.

关 键 词：酰胺化 核多角体病毒 活性表达 PHM 

分 类 号：Q78[生物学—分子生物学]