白纹伊蚊细胞色素P450 CYP6N3基因的原核表达及鉴定

目的　对白纹伊蚊细胞色素P4 5 0CYP6N3基因进行原核表达 ,以获得高效表达蛋白。方法　根据CYP6N3基因的全长cRNA为模板进行RT -PCR。产物经T -A克隆测序鉴定后 ,亚克隆入原核融合表达载体 pGEX - 4T - 1(含有编码 2 6KDaGST的基因序列 )中 ,在大肠杆菌BL2 1(DE3)中进行原核表达。将细菌总蛋白进行SDS聚丙烯酰胺凝胶电泳分析 ,通过Westernblot分析鉴定目的蛋白的位置 ,并运用核酸蛋白分析仪扫描凝胶以确定表达产物的含量。结果　获得了高效表达的融合蛋白GST -CYP6N3,表达量占菌体总蛋白的 37 4 9%。结论　本实验成功地异源表达了白纹伊蚊CYP6N3基因 ,为体外重建细胞色素P4 5 0CYP6N3单加氧酶系 ,了解CYP6N3基因结构功能关系奠定基础

Expression in Escherichia Coli and identification of a Cytochrome P450 gene CYP6N3 from Aedes albopictus

Aim Heterologous cytochrome P450 gene CYP6N3 from Aedes Albopictus was expressed in E.coli in order to get recombinant protein.Method According to the sequence of CYP6N3 cDNA,a pair of specific primers were designed.Total RNA was isolated from adults of Aedes albopictus deltamethrin resistant strain.CYP6N3 gene was amplified by RT PCR,cloned by T A ligation with T vector and identified by sequencing.Then,CYP6N3 was subcloned into the fusion expression vector pGEX 4T 1 (with GST encoding codons).The recombinant vectors were expressed in E.coli strain BL21.Total proteins of E.coli were analyzed by SDS PAGE and Western blot and scanned by nucleic acid protein analysis instrument.Results The fusion protein GST CYP6N3 was obtained,whose expression level was high up to 37.49% in total proteins of E.coli.Conclusion Through this study,CYP6N3 gene was expressed successfully in E.coli and the fusion protein GST CYP6N3 is obtained,which is a basis of reconstituting cytochrome P450 CYP6N3 monooxygenases and further learning structure function relationships of CYP6N3 gene.