Cloning and Expression Vector Construction of Mouse （Mus musculus） Bmal1 Gene and Its Bioinformatics Analysis

This study aims to clone the protein-coding sequence（CDS） of mouse（Mus musculus） circadian clock gene Bmal1, to construct a eukaryotic expression vector, and to further analyze the basic properties of the BMAL1 protein. Total RNA was extracted from the mouse liver, and the cDNA was obtained by reverse transcription PCR. The CDS fragments of Bmal1 gene were amplified by PCR, and then cloned into the pcDNA3.1-Puro-N-3 HA eukaryotic expression vector by homologous recombination. The plasmids were identified by restriction enzymes digestion and sequencing, and the positive recombinant plasmid was named pcDNA3.1-3 HA-BMAL1. After transfected into HEK293 T cells, the expression of Bmal1 at mRNA and protein levels was detected respectively by RT-qPCR and Western blot. Furthermore, bioinformatics analysis was carried out on the mouse Bmal1 gene and its encoded protein. The enzymatic identification and sequencing results indicated that the CDS region fragment of the Bmal1 gene inserted into the pcDNA3.1-3 HA-BMAL1 was correct. Compared with the control group transfected with pcDNA3.1-Puro-N-3 HA, the Bmal1 gene expression was significantly higher in HEK293 T cells transfected with pcDNA3.1-3 HA-BMAL1 vector at both mRNA and protein levels. Bioinformatics results showed that the Bmal1 gene had high homology in mammals, including humans and mice. The mouse BMAL1 protein, which composed primarily of 23 phosphorylation sites, 4 acetylation sites, and 6 ubiquitination sites, was somewhat hydrophilic but less stable. The mouse BMAL1 protein did not have the transmembrane domain and signal peptide as an intracellular protein. The secondary structure of BMAL1 consisted mainly of α-helix, extended chain, β-turn, and random coil, and the tertiary structure of mouse BMAL1 protein was similar to that of humans. The dual luciferase report experiment proved that the BMAL1 protein had normal transcriptional regulatory activity. In summary, the CDS fragment of the mouse Bmal1 gene was successfully cloned in this study, the mouse pcDNA3.1-3 HA-BMAL1 eukaryotic expression vector was constructed, its expression effects in HEK293 T cells were verified, and the basic properties of Bmal1 gene and its encoded protein were analyzed by bioinformatics softwares.