ATRA诱导上调基因RIG-C的cDNA克隆和序列分析及蛋白表达

Full-length cDNA cloning, sequence analysis and protein expression of retinoic-acid induced gene C

目的 克隆和表达维甲酸诱导上调的新基因RIG-C。方法 运用Blast、pfam等生物信息学手段对RIG-C的全长cDNA序列进行结构与功能预测;将RIG-C全长阅读框架插入原核表达载体pET22b中,在JM109(DE3)菌株中进行诱导表达;将全长RIG-C插入真核表达的绿荧光蛋白载体pEGFP-C1中,转染NIH3T3细胞以观察RIG-C的亚细胞定位。结果 RIG-C基因cDNA全长1266 bp,编码421个氨基酸,N端具有多个WD40结构域,C端有一个SOCS家族典型结构。原核表达的RIG-C蛋白大小约45 kd,NIH 3T3细胞表达的RIG-C定位于细胞浆。结论 克隆和表达了新基因RIG-C,为进一步研究其生物学功能和蛋白质的空间结构奠定了基础。

Objective The aim of this study is to clone and express the retinoicacid induced new gene C ( RIG-C). Methods The full-length cDNA sequence of RIG-C was analyzed with different bioinformatic tools such as blast, pfam etc. The ORF of RIG-C was cloned into pET22b vector, and induced expression in JM109 ( DE3 ) strain. At the same time, the ORF was cloned into green fluorescence protein vector pEGFP-Cl to study the location of RIG-C in the mammalian NIH 3T3 cells. Results The full-length cDNA of RIG-C was 1266 bp and encoding 421 aa peptide. Bioinformatical analysis showed that there were several WD40 domains in the N terminus, and one SOCS family domain in the C-terminus. The RIG-C encodes a 45 kd protein by prokaryotic vector in E coli. The location of RIG-C in the NIH 3T3 cells is cytoplasm. Conclusion The cloning and expression of RIG-C are basis of studying its biological function and crystal structure.