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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。昆虫细胞中表达的ＰＨＭ可直接用于甘氨肽的酰胺化修饰
The gene encoding rat glycoprotein α-hydroxylase (PHM) cDNA was inserted into the insect baculovirus transfer expression vector pBacPAK8 to construct the expression plasmid pBacPHM2, which was then ligated with the modified Spodoptera exigua polyhedrosis virus BacPAK6 DNA was co-transfected with Sf21, and the recombinant baculovirus BacPHM was obtained by homologous recombination to obtain the PHM gene under the control of nucleocapsid protein gene promoter. Sf21 cells were infected with BacPHM, and the highest activity of amidase was detected after 72 hours in serum-free culture supernatant. The expression products were detected by immunohistochemistry and immunoblotting. Both the intracellular and extracellular culture supernatants showed About 41kD of PHM expression product, intracellular and extracellular yields were about 5μg / 105 cells, 1μg / ml medium / 105 cells, and the secretion of the enzyme activity in the medium is much higher than intracellular. The PHM expressed in insect cells can be used directly for the amidation of glycyl peptides