胆固醇氧化酶基因的克隆及在E.coli中的表达

根据NCBI中报道的BrevibacteriumsterolicumATCC21387胆固醇氧化酶基因序列,采用PCR方法以Brevibacteriumsp.DGCDC-82的基因组为模板,扩增得到了编码胆固醇氧化酶的基因,该基因与来源于BrevibacteriumsterolicumATCC21387的胆固醇氧化酶基因(choB)同源性为98%。将得到的基因定向克隆到pET28a载体中,转化至含有编码argU和proL基因的大肠杆菌BL21-CodonPlus(DE3)-RP中表达。经过IPTG诱导后,经SDS-PAGE检测在约55kD处有一蛋白表达条带,目的蛋白表达量约占总蛋白的16%,经测定酶活为340U/L。

Cloning and Expression of Brevibacterium sp. DGCDC-82 Cholesterol Oxidase Gene in Escherichia coli

Based on DNA sequence encoding cholesterol oxidase reported on the NCBI,cholesterol oxidase gene was cloned from Brevibacterium sp. DGCDC-82 by PCR methods,which showed homology of 98% to the previously reported cholesterol oxidase gene from Brevibacterium sterolicum ATCC21387. Subsequently,the resulting products were digested with NcoI and EcoRI and ligated to the pET28a vector by T4 DNA ligase.The recombinant plasmid,pET28a-choB,was transformed into Escheriehia coli BL21-CodonPlus(DE3)-RP which contain extra copies of the argU and proL genes.The positive clone was induced with IPTG,and enzyme expressed in BL21-CodonPlus(DE3)-RP,the enzyme activity was about 340U/L.The expression products were analyzed by SDS-polyacrylamide gel electrophoresis indicating that about 55kD protein was obtained,which accounted for about 16% of the total cell protein.