鸭CYP7α1基因原核表达载体的构建及蛋白诱导表达

Construction of Prokaryotic Expression Vector containing CYP7 alpha 1 Gene of Duck and the Expression of Induced Protein

本研究旨在构建鸭重组表达载体pET32a-CYP7α1,获取其融合蛋白,为开展鸭CYP7α1蛋白在脂类代谢中作用的深入研究提供基础。试验以樱桃谷鸭为材料,通过克隆获得CYP7α1基因的ORF序列,并连接至原核表达载体pET32a中,构建了重组表达载体pET32a-CYP7α1,转入表达宿主Rossetta-gam(iDE3)进行诱导表达。结果表明CYP7α1基因ORF区共编码512个氨基酸,SDS-PAGE电泳分析表明,重组蛋白大小约为59 ku,其主要以包涵体的形式存在,其最佳诱导条件为温度37℃,时间4 h,IPTG浓度0.6 mmol/L。

This study was carried out to construct the duck recombinant expression vector to obtain the fusion protein to provide the basis for further researching the role of CYP7 alpha 1 protein in lipid metabolism. Taking Cherry Valley duck as the material in this study, the ORF of CYP7 alpha 1 gene of duck by cloning was inserted into pET32a prokaryotie expression vector. The recombinant plasmid named pET32a-CYP7a1 was constructed, and secondly transformed into Rossetta-gami（DE3）eells. SDS-PAGE analysis showed that the ORF region of CYP7 alpha 1 gene encodes 512 amino acidstotally, the recombinant protein weighed about 59 ku mainly existed in the form of inclusion body, and the optimum condition for induced exoression was 37℃ .4 h. 0.6 mmol/L IPTG