红鳍东方鲀(Takifugu rubripes)干扰素调节因子2基因的克隆及原核表达

Cloning and Prokaryotic Expression of Interferon Regulatory Factor 2 from Takifugu rubripes

干扰素调节因子(Interferon regulatory factor,IRF)是一种多功能的转录因子。采用反转录聚合酶链式反应(RT-PCR)和c DNA末端快速扩增(RACE)技术克隆了红鳍东方鲀干扰素调节因子2(Interferon regulatory factor 2,IRF2)基因的全长c DNA。该基因全长为1 552 bp,其中5'非编码区(5'-UTR)187 bp,3'非编码区(3'-UTR)429 bp,编码区(CDS)为936 bp,共编码311个氨基酸。将IRF2的编码区序列连接到原核表达载体p ET32a(+),成功构建了重组体p ET32a(+)-IRF2。将重组体p ET32a(+)-IRF2转入大肠杆菌感受态细胞BL21(DE3)中,获得了重组表达IRF2的基因工程菌。经IPTG诱导,p ET32a(+)-IRF2在BL21中得到了融合表达。通过对表达产物的纯化和Western blotting检测,结果显示红鳍东方鲀IRF2基因在大肠杆菌中的表达效果较高,目的蛋白准确。

Interferon Regulatory Factor（IRF）is a transcription factor with multifunctions. The full length c DNA of Takifugu rubripes interferon regulatory factor 2（IRF2）was cloned by RT-PCR and rapid amplification of c DNA ends（RACE）. The full-length c DNA of the gene was 1 552 bp,including a 187 bp 5＇ non-coding region,a 429 bp 3＇ non-coding region,and 936 bp coding region,and it encoded 311 amino acids. The recombinant p ET32a（＋）-IRF2 was constructed by ligating code sequence of IRF2 with prokaryotic expression vector p ET32a（＋）. Then,the recombinant genetic engineering bacteria with expressed IRF2 were obtained by transforming p ET32a（＋）-IRF2 into competent cell Escherichia coli BL21（DE3）. p ET32a（＋）-IRF2 was expressed in BL21 under the induction of IPTG. The results from the detection of the purified products by Western blotting showed that the expression of the IRF2 gene in E. coli was high,and the target protein was accurate.