GL-7-ACA酰化酶基因克隆及在大肠杆菌中的表达  

Cloning of GL-7-ACA Acylase Gene from Pseudomonas sp. 130 and Its Expression in Escherichia coli

作　　者：杨蕴刘[1] 恽定芳 关颖谦 彭惠林 陈剑民 何云生 焦瑞身[1] 

机构地区：[1]中国科学院上海植物生理研究所

出　　处：《生物工程学报》1991年第2期99-107,共9页Chinese Journal of Biotechnology

摘　　要：本文报道了利用BamHI酶解的载体pBR322 DNA与Sau3A部分酶解的假单胞菌Ps.130染色体DNA片段构建重组质粒,并用^(32)P标记的寡核苷酸探针从3205个含有外源片段的重组质粒中检测得到7株阳性克隆株。进一步经^(125)I一标记的抗体/抗原放射免疫反应、GL-7-ACA酰化酶活力测定以及酶反应产物的纸谱色层分析,由上述7株阳性株中鉴定出3株能在大肠杆菌中表达酰化酶活力的基因克隆株。对该3株菌的重组质粒——pMR 5、pMR 6和pMR7 DNA的初步凝胶电泳分析表明,pMR 5和pMR 7质粒中插入片段的分子量为6.8kb,质粒pMR 6则带有5.7kb的外源片段。实验还比较了重级质粒pMR5在8株不同的大肠杆菌宿主菌中,GL-7-ACA酰化酶的表达水平。Using BamHI digested and dephosphorylated pBR322 as vector, aGL-7-ACA acylase gene from Pseudomonas sp. 130 chromosomal DNA wascloned and expressed in E. coli C600. Seven positiive clones weredetected from 3205 recombinant plasmids with ^(32)-labeled oligonucleotidin situ hybridization. Out of them, three clones which produce activeGL-7-ACA acylase were identified by radio-immunological assay, che-mical test and chromatographic analysis of reaction minture.The plasmidDNA of recombinant pMR5, pMR6 and pMR7 were extracted. Analysisof gel electrophoresis indicated that pMR5 and pMR7 contained the same 6.8kb fragment and the size of the insert in pMR6 was 5.7kb.Theeffect of various E.coli hosts on the expression of cloned CL-7-ACAacylase gene is also reported.

关 键 词：7-ACA酰化酶 基因克隆 基因表达 

分 类 号：Q78[生物学—分子生物学]