人细胞色素CYP2E1基因重组杆状病毒粘粒构建

Cloning and identification of human CYP2E1 cDNA and construction of its recombinant bacmid DNA for baculovirus expression system

目的构建人细胞色素P450 2E1(CYP2E1)基因cDNA重组的杆状病毒粘粒。方法采用PCR方法,从国外获赠的含有CYP2E1基因cDNA的质粒中扩增CYP2E1 cDNA。将该基因的cDNA连接到测序载体pCMV-Myc载体上,通过内切酶分析及测序加以鉴定,然后将该基因的cDNA克隆到转座载体pFASTBac1载体,转化入DH10Bac大肠埃希菌感受态细胞中,提取含有CYP2E1基因的重组杆状病毒粘粒,并经PCR鉴定分析。结果测序结果显示,该克隆片段含有CYP2E1完整的编码区,所获得的重组杆状病毒粘粒含有完整1.5kb大小的该片段。结论获得重组CYP2E1杆状病毒粘粒,为下一步转染昆虫细胞,利用Bac-to-Bac杆状病毒表达系统大量表达人细胞色素P450 2E1蛋白打下基础。

Objective To obtain the recombinant bacmid with human cytochrome P450 2El （CYP2E1） cDNA suitable for expression in Bac - to - Bac baculovirus expression system, Methods Polymerase chain reaction（PCR） and DNA recom- binant techniques were used in the experiments to obtain CYP2E1 cDNA from an unknown plasmid with CYP2E1 gene and clone it into pCMV - Myc vector. The cDNA segment was analyzed by restriction enzyme digesting and DNA sequencing, Then the CYP2E1 cDNA was subcloned into vector pFastBac 1, and was transformed into competent E. coli DHIOBac, generating the recombinant bacmid. Results The gene segment was confirmed to be the whole coding region of CYP2E1 cDNA by DNA sequencing analysis and the recombinant bacmid was confirmed to have the whole 1.5kb of CYP2E1 cDNA. Conclusion The recombinant bacmid with CYP2E1 cDNA was constructed for making preparation for the next expression of CYP2E1 in Bac - to - Bac baculovirus expression system.