大鼠甘氨肽酰化单氧酶基因的克隆与表达  

Cloning and Expression of the cDNA Coding for Rat Peptidylglycine α-Amidating Monooxygenase

作　　者：江智红[1] 杨毅[1] 屠红旻 杨新颖[1] 夏其昌[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：《生物化学与生物物理学报》1996年第6期606-615,共10页

基　　金：国家"863"高技术研究发展计划资助

摘　　要：本文采用原位杂交及ＰＣＲ方法，从大鼠脑ｃＤＮＡ库中，筛选到３个大鼠甘氨肽酰化单氧酶（ｒＰＡＭ）基因片段。经ＤＮＡ全序列分析表明，它们跨越ｒＰＡＭ－２全部编码区。通过点突变、ＰＣＲ重组技术等，分别拼接出此双功能酶的ｒＰＨＭ（氧化酶）、ｒＰＡＬ（裂解酶）及其ｒＰＡＭ全酶基因，并构建了多种大肠杆菌表达质粒。经温敏诱导。高效表达了ｒＰＡＭ－Ｎ２６０片段，并制各获得阳性抗血清，可用于免疫检测天然或重组的ＰＡＭ。而ＳＤＳ－ＰＡＧＥ和Ｗｅｓｔｅｒｎｂｌｏｔ分析结果显示，ｒＰＨＭ和ｒＰＡＭ在大肠杆菌中均获得了表达，其中ｒＰＨＭ表达量占全菌总蛋白的１０％以上。进一步研究还发现，通过采用低温和加二价铜离子诱导表达，可提高ｒＰＨＭ产物的可溶性及稳定性。The cDNA of genes encoding rat peptidylglycine α-aminating monooxygenase (rPAM) were cloned and their expression in E.coli studied.Three DNA fragments were isolated from the rat brain cDNA library using the methods of plaque hybridization and PCR.DNA sequencing showed that they contained the total coding sequence for rPAM-2.By using the ding-directed mutation and PCR recombination methods,we obtained intact genes coding for the rPHM domain,the rPAL domain and reAM,respectively. Different plasmids of these genes controlled under T7 or PL promoter were constructed and transformed into E.coli.The high-level expression of rPAM-N260 in E.coli was first obaerved and its antiserum was prepared for the immunoassay of natural or recombinant PAMs.By the analysis with SDS-PAGE and Western blot,the Products of rPHM and rPAM in E. coli were detected,and the amount of rPHM reached over 10% of the total baoterial proteins.It was found that low temperature and copper ion obviously increased the stability and solubility of the rPHM expressed in E. coli.

关 键 词：甘氨肽化 单氧酶 氧化酶 裂解酶 基因表达 

分 类 号：Q554[生物学—生物化学]